Targeted integration of nucleic acids

ABSTRACT

The presently disclosed subject matter relates to targeted integration (TI) host cells suitable for the expression of recombinant proteins, as well as methods of producing and using said TI host cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/609,806, filed Dec. 22, 2017, and U.S. Provisional Application No.62/711,272, filed Jul. 27, 2018, the disclosures of each of which areincorporated herein by reference in its entirety.

TECHNICAL FIELD

The presently disclosed subject matter relates to targeted integration(TI) host cells suitable for the expression of recombinant proteins, aswell as methods of producing and using said TI host cells.

SEQUENCE LISTING

The present specification makes reference to a Sequence Listing(submitted electronically as a .txt file named “00B2060779SEQ.txt” onDec. 21, 2018). The 00B2060779SEQ.txt file was generated on Dec. 20,2018 and is 1,251,240 bytes in size. The entire contents of the SequenceListing are hereby incorporated by reference.

BACKGROUND

Due to the rapid advancement in cell biology and immunology, there hasbeen an increasing demand to develop novel therapeutic recombinantproteins for a variety of diseases including cancer, cardiovasculardiseases and metabolic diseases. These biopharmaceutical candidates arecommonly manufactured by commercial cell lines capable of expressing theproteins of interest. For example, Chinese hamster ovary (CHO) cellshave been widely adapted to produce monoclonal antibodies.

The conventional strategy for developing a commercial cell line involvesthe random integration of a nucleotide sequence encoding the polypeptideof interest followed by selection and isolation of cell lines producingthe polypeptide of interest. This approach, however, has severaldisadvantages. First, such integration is not only a rare event but,given the randomness as to where the nucleotide sequence integrates,these rare events can result in a variety of gene expression and cellgrowth phenotypes. Such variation, known as “position effect variation,”originates, at least in part, from the complex gene regulatory networkspresent in eukaryotic cell genomes and the accessibility of certaingenomic loci for integration and gene expression. Second, randomintegration strategies generally do not offer control over the number ofgene copies integrated into a host cell genome. In fact, geneamplification methods are often used to achieve high-producing cells.Such gene amplification, however, can lead to unwanted cell phenotypessuch as unstable cell growth and/or product expression. Third, becauseof the integration loci heterogeneity inherent in the random integrationprocess, it is time-consuming and labor-intensive to screen thousands ofclones after transfection to isolate cell lines demonstrating adesirable level of expression of the polypeptides of interest. Evenafter isolating such cell lines, stable expression of the polypeptide ofinterest is not guaranteed and further screening may be required toobtain a stable commercial cell line. Finally, polypeptides producedfrom randomly integrated cell lines exhibit a high degree of sequencevariance, which may be, in part, due to the mutagenicity of theselective agents used to select for a high level of expression ofpolypeptides of interest.

SUMMARY OF THE INVENTION

The presently disclosed subject matter relates to targeted integration(TI) host cells suitable for the expression of recombinant proteins. Thepresently disclosed subject matter not only provides host cell TI sitesthat have high productivity, it also provides a novel method ofintroducing multiple sequences of interest into a single TI locus in ahost cell by recombinase-mediated cassette exchange (RMCE). Theadvantages of the RMCE method include increased productivity and theflexibility to co-express multiple polypeptides in varying ratios fromthe same TI locus. For example, but not by way of limitation, the RMCEstrategies disclosed herein allow for the insertion of one, two, three,four, five, six, seven, eight or more sequences (e.g., antibody heavychain (HC) or light chain (LC) sequences) to the TI locus. With theability to target eight or more sequences simultaneously, thetwo-plasmid RMCE enables modulation of HC and LC chain ratios of mAb toimprove productivity, expression of complex molecules with multiplechains, and targeting of transgenes, endogenous genes or RNAi with theantibody to modify cellular pathways.

In certain embodiments, a TI host cell comprises an exogenous nucleotidesequence integrated at an integration site within a locus of the genomeof the host cell, wherein the locus is at least about 90% homologous toa sequence selected from SEQ ID Nos. 1-7. In certain embodiments, thenucleotide sequence immediately 5′ of the integrated exogenousnucleotide sequence is selected from the group consisting of sequencesat least about 90% homologous to nucleotides 41190-45269 ofNW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides253831-491909 of NW_006881296.1, nucleotides 69303-79768 ofNW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 ofNW_003615411.1. In certain embodiments, the nucleotide sequenceimmediately 3′ of the integrated exogenous nucleotide sequence isselected from the group consisting of sequences at least about 90%homologous to nucleotides 45270-45490 of NW_006874047.1, nucleotides207912-792374 of NW_006884592.1, nucleotides 491910-667813 ofNW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 ofNW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1. Incertain embodiments, the integrated exogenous nucleotide sequence isoperably linked to a nucleotide sequence selected from the groupconsisting of sequences at least about 90% homologous to a sequenceselected from SEQ ID Nos. 1-7.

In certain embodiments, a TI host cell comprises an exogenous nucleotidesequence integrated at an integration site within an endogenous geneselected from the group consisting of LOC107977062 (SEQ ID NO. 1),LOC100768845 (SEQ ID NO. 2), ITPR2 (SEQ ID NO. 3), ERE67000.1 (SEQ IDNO. 4), UBAP2 (SEQ ID NO. 5), MTMR2 (SEQ ID NO. 6), XP_003512331.2 (SEQID NO. 7), and sequences at least about 90% homologous thereto. Incertain embodiments, the exogenous nucleotide sequence is integrated atan integration site operably linked to an endogenous gene selected fromthe group consisting of LOC107977062 (SEQ ID NO. 1), LOC100768845 (SEQID NO. 2), ITPR2 (SEQ ID NO. 3), ERE67000.1 (SEQ ID NO. 4), UBAP2 (SEQID NO. 5), MTMR2 (SEQ ID NO. 6), XP_003512331.2 (SEQ ID NO. 7), andsequences at least about 90% homologous thereto. In certain embodiments,the integrated exogenous nucleotide is flanked by a nucleotide sequenceselected from the group consisting of sequences at least about 90%homologous to a sequence selected from SEQ ID Nos. 1-7. In certainembodiments, the exogenous nucleotide sequence is integrated at anintegration site immediately adjacent to all or a portion of a sequenceselected from the group consisting of sequences at least about 90%homologous to a sequence selected from SEQ ID Nos. 1-7.

In certain embodiments, a TI host cell is a mammalian host cell. Incertain embodiments, a TI host cell is a hamster host cell, a human hostcell, a rat host cell, or a mouse host cell. In certain embodiments, aTI host cell is a Chinese hamster ovary (CHO) host cell, a CHO K1 hostcell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 host cell, aCHOK1S host cell, or a CHO KIM host cell.

In certain embodiments, a TI host cell comprises an integrated exogenousnucleotide sequence, wherein the exogenous nucleotide sequence comprisesone or more recombination recognition sequence (RRS). In certainembodiments, the exogenous nucleotide sequence comprises at least twoRRSs. The RRS can be recognized by a recombinase, for example, a Crerecombinase, an FLP recombinase, a Bxb1 integrase, or a φC31 integrase.The RRS can be selected from the group consisting of a LoxP sequence, aLoxP L3 sequence, a LoxP 2 L sequence, a LoxFas sequence, a Lox511sequence, a Lox2272 sequence, a Lox2372 sequence, a Lox5171 sequence, aLoxm2 sequence, a Lox71 sequence, a Lox66 sequence, a FRT sequence, aBxb1 attP sequence, a Bxb1 attB sequence, a φC31 attP sequence, and aφC31 attB sequence.

In certain embodiments, the exogenous nucleotide sequence comprises afirst and a second RRS, and at least one selection marker locatedbetween the first and the second RRS. In certain embodiments, theexogenous nucleotide sequence further comprises a third RRS, wherein thethird RRS is located between the first and the second RRS, and the thirdRRS is different from the first or the second RRS. In certainembodiments, the exogenous nucleotide sequence comprises a first and asecond RRS, and a first selection marker located between the first andthe second RRS. In certain embodiments, the exogenous nucleotidesequence further comprises a second selection marker, and the first andthe second selection markers are different. In certain embodiments, theexogenous nucleotide sequence can further comprise a third selectionmarker and an internal ribosome entry site (IRES), wherein the IRES isoperably linked to the third selection marker. The third selectionmarker can be different from the first or the second selection marker.The selection markers can be selected from the group consisting of anaminoglycoside phosphotransferase (APH) (e.g., hygromycinphosphotransferase (HYG), neomycin and G418 APH), dihydrofolatereductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS),asparagine synthetase, tryptophan synthetase (indole), histidinoldehydrogenase (histidinol D), and genes encoding resistance topuromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin,and mycophenolic acid. The selection markers can also be selected fromthe group consisting of a green fluorescent protein (GFP) marker, anenhanced GFP (eGFP) marker, a synthetic GFP marker, a yellow fluorescentprotein (YFP) marker, an enhanced YFP (eYFP) marker, a cyan fluorescentprotein (CFP) marker, an mPlum marker, an mCherry marker, a tdTomatomarker, an mStrawberry marker, a J-red marker, a DsRed-monomer marker,an mOrange marker, an mKO marker, an mCitrine marker, a Venus marker, aYPet marker, an Emerald6 marker, a CyPet marker, an mCFPm marker, aCerulean marker, and a T-Sapphire marker. In some embodiments, theselection marker is selected from the group consisting of a greenfluorescent protein (GFP) marker, an enhanced GFP (eGFP) marker, asynthetic GFP marker.

In certain embodiments, the exogenous nucleotide sequence comprises atleast one selection marker and at least one exogenous sequence ofinterest (SOI). In certain embodiments, the exogenous nucleotidesequence comprises a first and a second RRS, and at least one selectionmarker and at least one exogenous SOI located between the first and thesecond RRS. In certain embodiments, the exogenous nucleotide sequencecomprises a first, second, and third RRS, at least one selection markerand at least one exogenous SOI located between the first and the thirdRRS, and at least one exogenous SOI located between the third and thesecond RRS. The sequence of interest can encode any polypeptide ofinterest, including but not limited to the examples listed herein. TheSOIs can be the same or they can be different, for example the SOIs caninclude coding sequences to both chains of an antibody. In the casewhere two or more different polypeptides are expressed, the sequence ofinterest can include the two polypeptides at various ratios, e.g., wheneight coding sequences encode two different polypeptides including, butnot limited to, 1:7, 2:6, etc. The SOIs can encode a single chainantibody, an antibody light chain, an antibody heavy chain, asingle-chain Fv fragment (scFv), or an Fc fusion protein.

The presently disclosed subject matter also provides methods for thetargeted integration of an exogenous nucleic acid into a host cell tofacilitate the expression of a polypeptide of interest. In certainembodiments, such methods comprise the targeted integration of anexogenous nucleic acid into a host cell via recombinase-mediatedrecombination. In certain embodiments, such methods relate to a cellcomprising an exogenous nucleotide sequence integrated at a site withinan endogenous gene selected from the group consisting of LOC107977062,LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, andsequences least about 90% homologous thereto. In certain embodiments,such methods relate to a cell comprising an exogenous nucleotidesequence integrated within a locus of the genome of the host cell,wherein the locus comprises a nucleotide sequence that is at least about90% homologous to a sequence selected from SEQ ID Nos. 1-7.

In certain embodiments, the present disclosure provides a method ofpreparing a TI host cell expressing a polypeptide of interest comprises:a) providing a TI host cell comprising an exogenous nucleotide sequenceintegrated at a site within a locus of the genome of the TI host cell,wherein the locus is at least about 90% homologous to a sequenceselected from SEQ ID Nos. 1-7, wherein the exogenous nucleotide sequencecomprises two RRSs, flanking at least one first selection marker; b)introducing into the cell provided in a) a vector comprising two RRSsmatching the two RRSs on the integrated exogenous nucleotide sequenceand flanking at least one exogenous SOI and at least one secondselection marker; c) introducing a recombinase, wherein the recombinaserecognizes the RRSs; and d) selecting for TI cells expressing the secondselection marker to thereby isolate a TI host cell expressing thepolypeptide of interest.

In certain embodiments, the present disclosure provides a method ofpreparing a TI host cell expressing a first and second polypeptide ofinterest (where the first and second polypeptides can be the same ordifferent) comprises: a) providing a TI host cell comprising anexogenous nucleotide sequence integrated at a site within a locus of thegenome of the host cell, wherein the locus is at least about 90%homologous to a sequence selected from SEQ ID Nos. 1-7, wherein theexogenous nucleotide sequence comprises a first and a second RRSflanking at least one first selection marker, and a third RRS locatedbetween the first and the second RRS, wherein the first, second, andthird RRSs are heterospecific, i.e., the first, second, and third RRSsare not matched RRSs and therefore any two of the first, second, andthird RRSs would not be amenable to recombinase-mediated recombination;b) introducing into the cell provided in a) a first vector comprisingtwo RRSs matching the first and the third RRS on the integratedexogenous nucleotide sequence and flanking at least one first exogenousSOI and at least one second selection marker; c) introducing into thecell provided in a) a second vector comprising two RRSs matching thesecond and the third RRS on the integrated exogenous nucleotide sequenceand flanking at least one second exogenous SOI; d) introducing one ormore recombinases, wherein the one or more recombinases recognize theRRSs; and e) selecting for TI cells expressing the second selectionmarker to thereby isolate a TI host cell expressing the first and secondpolypeptides of interest.

In certain embodiments, the present disclosure provides a method forexpressing a polypeptide of interest comprises: a) providing a host cellcomprising at least one exogenous SOI and at least one selection markerflanked by two RRSs integrated within a locus of the genome of the hostcell, wherein the locus is at least about 90% homologous to a sequenceselected from SEQ ID Nos. 1-7; and b) culturing the cell in a) underconditions suitable for expressing the SOI and recovering a polypeptideof interest therefrom.

In certain embodiments, the present disclosure provides a method forexpressing a polypeptide of interest comprises: a) providing a host cellcomprising at least two exogenous SOIs and at least one selection markerintegrated within a locus of the genome of the host cell, wherein thelocus is at least about 90% homologous to a sequence selected from SEQID Nos. 1-7, wherein at least one exogenous SOI and one selection markeris flanked by a first and a third RRS and at least one exogenous SOI isflanked by a second and the third RRS; and b) culturing the cell in a)under conditions suitable for expressing the SOI and recovering apolypeptide of interest therefrom.

The presently disclosed subject matter also provides compositions andmethods to produce polypeptides of interest via the use of TI hostcells. Such compositions and methods include, but are not limited to,polynucleotides and vectors, facilitating the integration of anexogenous nucleotide sequence into a TI host cell as well as TI hostcells and compositions comprising exogenous nucleotide sequences andmethods of using the same. In certain embodiments, a vector can comprisenucleotide sequences at least 50% homologous to a sequence selected fromSEQ ID Nos. 1-7 flanking a DNA cassette, wherein the DNA cassettecomprises at least one selection marker and at least one exogenous SOIflanked by two RRSs. The vector can be selected from the groupconsisting of an adenovirus vector, an adeno-associated virus vector, alentivirus vector, a retrovirus vector, an integrating phage vector, anon-viral vector, a transposon and/or transposase vector, an integrasesubstrate, and a plasmid.

In certain embodiments, the methods of the present disclosure comprisethe targeted integration of an exogenous nucleic acid encoding apolypeptide of interest into a host cell via homologous recombination,homology-directed repair (HDR), and/or non-homologous end joining(NHEJ). In certain embodiments, such methods involve the integration ofan exogenous nucleic acid encoding a polypeptide of interest into a cellat a site within an endogenous gene selected from the group consistingof LOC107977062 (SEQ ID NO. 1), LOC100768845 (SEQ ID NO. 2), ITPR2 (SEQID NO. 3), ERE67000.1 (SEQ ID NO. 4), UBAP2 (SEQ ID NO. 5), MTMR2 (SEQID NO. 6), XP_003512331.2 (SEQ ID NO. 7), and sequences at least about90% homologous thereto. In certain embodiments, such methods involve theintegration of an exogenous nucleic acid encoding a polypeptide ofinterest into a cell at a site within a locus of the genome of the hostcell, wherein the locus comprises a nucleotide sequence that is at leastabout 90% homologous to a sequence selected from SEQ ID Nos. 1-7.

In certain embodiments, the present disclosure provides a method forpreparing a TI host cell expressing a polypeptide of interest comprises:a) providing a host cell comprising a locus of the genome of the hostcell, wherein the locus is at least about 90% homologous to a sequenceselected from SEQ ID Nos. 1-7; b) introducing a vector into the hostcell, wherein the vector comprises nucleotide sequences at least 50%homologous to a sequence selected from SEQ ID Nos. 1-7 flanking a DNAcassette, wherein the DNA cassette comprises at least one selectionmarker and at least one exogenous SOI flanked by two RRSs; and c)selecting for the selection marker to isolate a TI host cell with theSOI integrated in the locus of the genome, and expressing thepolypeptide of interest. In certain embodiments, such methods involvethe use of an exogenous nuclease to promote the targeted integration.The exogenous nuclease can be selected from the group consisting of azinc finger nuclease (ZFN), a ZFN dimer, a transcription activator-likeeffector nuclease (TALEN), a TAL effector domain fusion protein, anRNA-guided DNA endonuclease, an engineered meganuclease, and a clusteredregularly interspaced short palindromic repeats (CRISPR)-associated(Cas) endonuclease.

In certain embodiments, the present disclosure provides a method ofpreparing a TI host cell expressing a polypeptide of interestcomprising: a) providing a TI host cell comprising at least oneexogenous nucleotide sequence integrated at a site within one or moreloci of the genome of the TI host cell, wherein the one or more loci areat least about 90% homologous to a sequence selected from SEQ ID No.1-7, wherein the exogenous nucleotide sequence comprises one or moreRRSs; b) introducing into the cell provided in a) a vector comprisingone or more RRSs matching the one or more RRSs on the integratedexogenous nucleotide sequence and flanking at least one exogenous SOIoperably linked to a regulatable promoter; c) introducing a recombinaseor a nucleic acid encoding a recombinase, wherein the recombinaserecognizes the RRSs; and d) selecting for TI cells expressing theexogenous SOI in the presence of an inducer to thereby isolate a TI hostcell expressing the polypeptide of interest.

In certain embodiments, the present disclosure provides a method forexpressing a polypeptide of interest comprising: a) providing a hostcell comprising at least one exogenous SOI flanked by two RRSs andoperably linked to a regulatable promoter integrated within a locus ofthe genome of the host cell, wherein the locus is at least about 90%homologous to a sequence selected from SEQ ID Nos. 1-7; and b) culturingthe cell under conditions suitable for expressing the SOI and recoveringa polypeptide of interest therefrom.

In certain embodiments, the present disclosure provides a method forpreparing a TI host cell expressing a first and second polypeptide ofinterest (where the first and second polypeptides can be the same ordifferent) comprising: a) providing a TI host cell comprising anexogenous nucleotide sequence integrated at a site within a locus of thegenome of the host cell, wherein the locus is at least about 90%homologous to a sequence selected from SEQ ID Nos. 1-7, wherein theexogenous nucleotide sequence comprises a first, second RRS and a thirdRRS located between the first and the second RRS, wherein the first,second, and third RRSs are heterospecific, i.e. the first, second, andthird RRSs are not matched RRSs and therefore any two of the first,second, and third RRSs would not be amenable to recombinase-mediatedrecombination; b) introducing into the cell provided in a) a firstvector comprising two RRSs matching the first and the third RRS on theintegrated exogenous nucleotide sequence and flanking at least one firstexogenous SOI operably linked to a regulatable promoter; c) introducinginto the cell provided in a) a second vector comprising two RRSsmatching the second and the third RRS on the integrated exogenousnucleotide sequence and flanking at least one second exogenous SOIoperably linked to a regulatable promoter; d) introducing one or morerecombinases, or one or more nucleic acids encoding one or morerecombinases, wherein the one or more recombinases recognize the RRSs;and e) selecting for TI cells expressing the exogenous SOI in thepresence of an inducer to thereby isolate a TI host cell expressing thefirst and second polypeptides of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing an outline of the genome-wide screeningsteps for identifying CHO TI loci that allow for stable and highexpression of antibodies.

FIGS. 2A and 2B depict two double selection schemes which were utilizedto generate the targeted antibody expressing population. Transposasegenerated TI hosts are depicted in FIG. 2A and random integrationgenerated TI hosts are depicted in FIG. 2B.

FIG. 3 depicts the productivity of the top clones generated by twoparticular TI hosts.

FIG. 4 depicts a two-plasmid RMCE strategy involving the use of threeRRS sites to carry out two independent RMCEs simultaneously. One vector(front) comprises the RRS1, a first SOI and a promoter (P) followed by astart codon (ATG) and RRS3. The other vector (back) comprises the RRS3fused to the coding sequence of a marker without the start codon (ATG),an SOI 2 and the RRS2. Only when the two plasmids are correctly targetedwill the full expression cassette of the selection marker be assembledthus rendering cells resistance to selection.

FIG. 5 depicts the results of using either single-vector or two-vectorRMCE in connection with the expression of Antibodies D, E, F, G, and J.FACS, genomic PCR, and gene copy analysis were assessed to confirm bothplasmid cassettes were targeted correctly to the TI site. In two-plasmidRMCEs, more total HC and LC copies were targeted to the TI site ascompared to single plasmid RMCE.

FIG. 6 depicts the titers of six molecules.

FIG. 7 depicts the results of using two-vector RMCE to express complexmAb formats. Two bispecific molecules, each requiring four differentchains (two HCs and two LCs), were assay for production and % bispecificformation. In both cases, cell lines were developed exhibiting >1.5 g/Lwith >80% bispecific content.

FIG. 8 depicts the day 14 titers of four bispecific molecules.

FIGS. 9 A and B depict the generation of cell lines expressing mAb-I ormAb-II in the RTI system. Histological results for cell line developmentare depicted in FIG. 9A and a schematic of a RTI cell line development(CLD) process is depicted in FIG. 9B.

FIGS. 10 A to C depict the titer and specific productivity results ofcell lines expressing mAb-I or mAb-II. Production end titer is depictedin FIG. 10A, production end IVCC is depicted in FIG. 10B and productionend specific productivity is depicted in FIG. 10C.

FIGS. 11 A and B depict the expression levels of HC and LC mRNA of mAb-Iand mAb-II expressing cell lines. The expression levels of HC mRNA aredepicted in FIG. 11A and the expression levels of HL mRNA are depictedin FIG. 11B.

FIGS. 12 A to D depict the intracellular levels of HC, HL and BiPmolecules in cell lines expressing mAb-I or mAb-II. A schematic ofcycloheximide and Dox effects on an RTI system is depicted in FIG. 12A.The intracellular levels of HC, LC and BiP as well as the HC and LClevels in the supernatants of cell lines expressing mAb-A or mAb-B aredepicted in FIG. 12B. The intracellular levels of HC, LC and BiP of celllines expressing mAb-I or mAb-II after overnight CHX treatment aredepicted in FIG. 12C. The intracellular levels of HC, LC and BiP of celllines expressing mAb-I or mAb-II after doxycycline removal from culturemedia are depicted in FIG. 12D.

FIGS. 13 A to D depict the correlation between the intracellularaccumulation of BiP and mAb-I LC expression. The production end titer isdepicted in FIG. 13A and the accumulation of HC, LC and BiP is depictedin FIG. 13B. A schematic representation of chain-swap experiments isdepicted in FIG. 13C. The intracellular levels of HC, LC and BiP of RTIpools expressing mAb-I, mAb-II, or chain-swapped versions of thesemolecules are depicted in FIG. 13D.

FIGS. 14 A to C depict the contribution of mAb-A HC and HL to the poolexpression of this molecule. Production end titer is depicted in FIG.14A, production end IVCC is depicted in FIG. 13B and production endspecific productivity is depicted in FIG. 14C.

FIGS. 15 A to B depict CDR regions of LC and HC that may contribute topoor expression. A schematic representation of the mAb-I and mAb-II LCsubunits is depicted in FIG. 15A. A schematic representation of mAb-Iand mAb-II HC regions is depicted in FIG. 15B.

FIGS. 16 A to C depict the results of using two point mutations in HCconstant region in connection with the expression of HCA. The productionend titer of various chain swaps between mAb-I and mAb-II HC and LCsubunits is depicted in FIG. 16A. The intracellular levels of HC, LC andBiP of various RTI pools are depicted in FIG. 16B. A schematic of RTIsystem utilization as a diagnostic tool for low protein expression isdepicted in FIG. 16C.

FIG. 17 depicts the production using two different cell cultureprocesses.

FIGS. 18 A to D depict the titer and specific productivity results forRMCE pools of three mAbs. Day 14 titer for RMCE pools of three mAbscomparing the HL and HLL configurations is depicted in FIG. 18A. Day 14specific productivity (Qp) for RMCE pools of three mAbs comparing the HLand HLL configurations is depicted in FIG. 18B. Day 14 titer for clonesgenerated from the pools of FIG. 18A are depicted in FIG. 18C. Day 14 Qpfor clones generated from the pools of FIG. 18A are depicted in FIG.18D. For FIGS. 18C and D six clones per configuration were tested.

FIGS. 19 A to F depict titer, Qp, and growth (as expressed by theintegral of viable cell concentration, IVCC), mRNA expression andprotein expression for RMCE pools of mAb Y comparing three differentplasmid configurations. Day 14 titer, Qp, and growth (as expressed bythe integral of viable cell concentration, IVCC) are depicted in FIGS.19 A, B and C respectively. Each bar represents data from a single pool.Seed train mRNA expression and intracellular antibody protein expressionfrom the pools in 19A. mRNA expression for heavy and light chain wasnormalized to the HLL-HL configuration; each bar the average of fourtechnical replicates (error bars are standard deviation) mRNA expressionfor heavy and light chain was normalized to the HLL-HL configuration.Intracellular heavy and light chain protein expression from all poolsquantified from western blots and normalized to the HLL-HLconfiguration, is depicted in FIG. 19E; each bar is the average of threetechnical replicates (error bars are standard deviation). Arepresentative western blot image of the data shown in FIG. 19E isdepicted in FIG. 19F.

FIGS. 20 A to F depict titer, Qp, and growth (as expressed by theintegral of viable cell concentration, IVCC) for mAb Y or mAb IIIexpressing monoclones generated from RMCE pools with three differentconfigurations. Day 14 titer for mAb Y expressing monoclones is depictedin FIG. 20A. Day 14 Qp for mAb Y expressing monoclones is depicted inFIG. 20B. Day 14 IVCC for mAb Y expressing clones is depicted in FIG.20C. Day 14 titer for mAb III expressing monoclones is depicted in FIG.20D. Day 14 Qp for mAb III expressing monoclones is depicted in FIG.20E. Day 14 IVCC for mAb III expressing clones is depicted in FIG. 20F.

FIGS. 21 A to D depict expression levels, titer and Qp of RMCE pools ofvarious configurations. A schematic of the one heavy chain, one lightchain plasmids transfected to assess the effect of plasmid position onchain expression is depicted in FIG. 21A. Antibody heavy (abbreviated asH) and light chain (abbreviated as L) were either expressed on the sameplasmid or split between the front and back plasmids. L3, Loxfas, and 2L sites are noted, as is thepac puromycin resistance gene in the backplasmid. Seed train mRNA expression of heavy and light chain from poolswith DNA configurations outlined in FIG. 21A are depicted in FIG. 23B.Day 14 titer for the RMCE pools of the various configurations isdepicted in FIG. 21C. Day 14 Qp of the various configurations isdepicted in FIG. 21D. Heavy and light chain DNA copy number for thepools is depicted in FIG. 21E.

FIG. 22 A to D depict that the effect of HC:LC ratio on theHigh-molecular-weight species (HMWS) percentage of mAbs is independentof the TI site. Day 14 titer for antibody S on Host 7 cells is depictedin FIG. 22A. High-molecular-weight species (HMWS) percentage of antibodyS is on Host 7 cells is depicted in FIG. 22B. Day 14 titer for antibodyS on Host 4 cells is depicted in FIG. 22C. High-molecular-weight species(HMWS) percentage of antibody S is on Host 4 cells is depicted in FIG.22D.

FIGS. 23 A and B depict the effect of HC:LC ration on productivity andHigh-molecular-weight species (HMWS) of antibody IV. Day 14 titer forantibody IV on Host 4 cells is depicted in FIG. 23A.High-molecular-weight species (HMWS) percentage of antibody IV on Host 4cells is depicted in FIG. 23D.

FIGS. 24 A and B depict the effect of HC:LC ratio on productivity andHigh-molecular-weight species (HMWS) of antibody VI in a two-site TIhost cells. Day 14 titer for antibody VI on two-site TI host cells isdepicted in FIG. 24A. High-molecular-weight species (HMWS) percentage ofantibody VI on two-site TI host cells is depicted in FIG. 24B.

FIG. 25 depicts 2 clones with titers of 8.4 and 8.7 g/L with 7.4% and5.6% aggregates, respectively.

FIG. 26 depicts Day 14 titer of antibody Z in different regulatedtargeted integration cell lines.

FIG. 27 specific productivity of antibody Z in different regulatedtargeted integration cell lines.

DETAILED DESCRIPTION

In certain embodiments, the host cells, genetic constructs (e.g.,vectors), compositions, and methods described herein can be employed inthe development and/or use of a targeted integration (TI) host cell. Incertain embodiments, such TI host cells comprise an exogenous nucleotidesequence integrated within a specific gene or a specific locus of thegenome of the host cell.

For purposes of clarity of disclosure and not by way of limitation, thedetailed description is divided into the following subsections:

1. Definitions

2. Integration Sites

3. Exogenous Nucleotide Sequences

4. Host Cells

5. Targeted Integration

6. Preparation and Use of TI Host Cells

7. Products

8. Exemplary Non-Limiting Embodiments

1. Definitions

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentlydisclosed subject matter. All publications, patent applications, patentsand other references mentioned herein are incorporated by reference intheir entirety. The materials, methods, and examples disclosed hereinare illustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “an” and “the” include plural references unless the context clearlydictates otherwise. The present disclosure also contemplates otherembodiments “comprising,” “consisting of”, and “consisting essentiallyof,” the embodiments or elements presented herein, whether explicitlyset forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

As used herein, the term “about” or “approximately” means within anacceptable error range for the particular value as determined by one ofordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system.For example, “about” can mean within 3 or more than 3 standarddeviations, per the practice in the art. Alternatively, “about” can meana range of up to 20%, preferably up to 10%, more preferably up to 5%,and more preferably still up to 1% of a given value. Alternatively,particularly with respect to biological systems or processes, the termcan mean within an order of magnitude, preferably within 5-fold, andmore preferably within 2-fold, of a value.

As used herein, the term “selection marker” can be a gene that allowscells carrying the gene to be specifically selected for or against, inthe presence of a corresponding selection agent. For example, but not byway of limitation, a selection marker can allow the host celltransformed with the selection marker gene to be positively selected forin the presence of the gene; a non-transformed host cell would not becapable of growing or surviving under the selective conditions.Selection markers can be positive, negative or bi-functional. Positiveselection markers can allow selection for cells carrying the marker,whereas negative selection markers can allow cells carrying the markerto be selectively eliminated. A selection marker can confer resistanceto a drug or compensate for a metabolic or catabolic defect in the hostcell. In prokaryotic cells, amongst others, genes conferring resistanceagainst ampicillin, tetracycline, kanamycin or chloramphenicol can beused. Resistance genes useful as selection markers in eukaryotic cellsinclude, but are not limited to, genes for aminoglycosidephosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG),neomycin and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase(TK), glutamine synthetase (GS), asparagine synthetase, tryptophansynthetase (indole), histidinol dehydrogenase (histidinol D), and genesencoding resistance to puromycin, blasticidin, bleomycin, phleomycin,chloramphenicol, Zeocin, and mycophenolic acid. Further marker genes aredescribed in WO 92/08796 and WO 94/28143.

Beyond facilitating a selection in the presence of a correspondingselection agent, a selection marker can alternatively provide a geneencoding a molecule normally not present in the cell, e.g., greenfluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellowfluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein(CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed-monomer,mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean,and T-Sapphire. Cells harboring such a gene can be distinguished fromcells not harboring this gene, e.g., by the detection of thefluorescence emitted by the encoded polypeptide.

As used herein, the term “operably linked” refers to a juxtaposition oftwo or more components, wherein the components are in a relationshippermitting them to function in their intended manner. For example, apromoter and/or an enhancer is operably linked to a coding sequence ifthe promoter and/or enhancer acts to modulate the transcription of thecoding sequence. In certain embodiments, DNA sequences that are“operably linked” are contiguous and adjacent on a single chromosome. Incertain embodiments, e.g., when it is necessary to join two proteinencoding regions, such as a secretory leader and a polypeptide, thesequences are contiguous, adjacent, and in the same reading frame. Incertain embodiments, an operably linked promoter is located upstream ofthe coding sequence and can be adjacent to it. In certain embodiments,e.g., with respect to enhancer sequences modulating the expression of acoding sequence, the two components can be operably linked although notadjacent. An enhancer is operably linked to a coding sequence if theenhancer increases transcription of the coding sequence. Operably linkedenhancers can be located upstream, within, or downstream of codingsequences and can be located a considerable distance from the promoterof the coding sequence. Operable linkage can be accomplished byrecombinant methods known in the art, e.g., using PCR methodology and/orby ligation at convenient restriction sites. If convenient restrictionsites do not exist, then synthetic oligonucleotide adaptors or linkerscan be used in accord with conventional practice. An internal ribosomalentry site (IRES) is operably linked to an open reading frame (ORF) ifit allows initiation of translation of the ORF at an internal locationin a 5′ end-independent manner.

As used herein, the term “expression” refers to transcription and/ortranslation. In certain embodiments, the level of transcription of adesired product can be determined based on the amount of correspondingmRNA that is present. For example, mRNA transcribed from a sequence ofinterest can be quantitated by PCR or by Northern hybridization. Incertain embodiments, protein encoded by a sequence of interest can bequantitated by various methods, e.g. by ELISA, by assaying for thebiological activity of the protein, or by employing assays that areindependent of such activity, such as Western blotting orradioimmunoassay, using antibodies that recognize and bind to theprotein.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), half antibodies, and antibody fragments so longas they exhibit a desired antigen-binding activity.

As used herein, the term “antibody fragment” refers to a molecule otherthan an intact antibody that comprises a portion of an intact antibodythat binds the antigen to which the intact antibody binds. Examples ofantibody fragments include but are not limited to Fv, Fab, Fab′,Fab′-SH, F(ab′)2; diabodies; linear antibodies; single-chain antibodymolecules (e.g., scFv); and multispecific antibodies formed fromantibody fragments. For a review of certain antibody fragments, seeHolliger and Hudson, Nature Biotechnology 23:1126-1136 (2005).

As used herein, the term “variable region” or “variable domain” refersto the domain of an antibody heavy or light chain that is involved inbinding the antibody to antigen. The variable domains of the heavy chainand light chain (V_(H) and V_(L), respectively) of a native antibodygenerally have similar structures, with each domain comprising fourconserved framework regions (FRs) and three hypervariable regions(HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freemanand Co., page 91 (2007).) A single V_(H) or V_(L) domain may besufficient to confer antigen-binding specificity. Furthermore,antibodies that bind to a particular antigen may be isolated using aV_(H) or V_(L) domain from an antibody that binds the antigen to screena library of complementary V_(L) or V_(H) domains, respectively. See,e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al.,Nature 352:624-628 (1991).

As used herein, the term “heavy chain” refers to an immunoglobulin heavychain.

As used herein, the term “light chain” refers to an immunoglobulin lightchain.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies in accordance with the present invention may bemade by a variety of techniques, including but not limited to thehybridoma method, recombinant DNA methods, phage-display methods, andmethods utilizing transgenic animals containing all or part of the humanimmunoglobulin loci, such methods and other exemplary methods for makingmonoclonal antibodies being described herein.

“Multispecific antibodies” are monoclonal antibodies that have bindingspecificities for at least two different sites, i.e., different epitopeson different antigens or different epitopes on the same antigen. Incertain aspects, the multispecific antibody has three or more bindingspecificities. Multispecific antibodies may be prepared as full-lengthantibodies or antibody fragments.

The terms “full length antibody”, “intact antibody”, and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv, and scFab); single domain antibodies (dAbs); andmultispecific antibodies formed from antibody fragments. For a review ofcertain antibody fragments, see Holliger and Hudson, NatureBiotechnology 23:1126-1136 (2005).

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human CDRs and amino acid residues from humanFRs. In certain aspects, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDRs correspond to those of anon-human antibody, and all or substantially all of the FRs correspondto those of a human antibody. A humanized antibody optionally maycomprise at least a portion of an antibody constant region derived froma human antibody. A “humanized form” of an antibody, e.g., a non-humanantibody, refers to an antibody that has undergone humanization. Theterm “monoclonal antibody” as used herein refers to an antibody obtainedfrom a population of substantially homogeneous antibodies, i.e., theindividual antibodies comprising the population are identical and/orbind the same epitope, except for possible variant antibodies, e.g.,containing naturally occurring mutations or arising during production ofa monoclonal antibody preparation, such variants generally being presentin minor amounts. In contrast to polyclonal antibody preparations, whichtypically include different antibodies directed against differentdeterminants (epitopes), each monoclonal antibody of a monoclonalantibody preparation is directed against a single determinant on anantigen. Thus, the modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method.

The term “therapeutic antibody” refers to an antibody that is used inthe treatment of disease. A therapeutic antibody may have variousmechanisms of action. A therapeutic antibody may bind and neutralize thenormal function of a target associated with an antigen. For example, amonoclonal antibody that blocks the activity of the of protein neededfor the survival of a cancer cell causes the cell's death. Anothertherapeutic monoclonal antibody may bind and activate the normalfunction of a target associated with an antigen. For example, amonoclonal antibody can bind to a protein on a cell and trigger anapoptosis signal. Yet another monoclonal antibody may bind to a targetantigen expressed only on diseased tissue; conjugation of a toxicpayload (effective agent), such as a chemotherapeutic or radioactiveagent, to the monoclonal antibody can create an agent for specificdelivery of the toxic payload to the diseased tissue, reducing harm tohealthy tissue. A “biologically functional fragment” of a therapeuticantibody will exhibit at least one if not some or all of the biologicalfunctions attributed to the intact antibody, the function comprising atleast specific binding to the target antigen.

The term “diagnostic antibody” refers to an antibody that is used as adiagnostic reagent for a disease. The diagnostic antibody may bind to atarget antigen that is specifically associated with, or shows increasedexpression in, a particular disease. The diagnostic antibody may beused, for example, to detect a target in a biological sample from apatient, or in diagnostic imaging of disease sites, such as tumors, in apatient. A “biologically functional fragment” of a diagnostic antibodywill exhibit at least one if not some or all of the biological functionsattributed to the intact antibody, the function comprising at leastspecific binding to the target antigen.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells”, which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

The term “nucleic acid molecule” or “polynucleotide” includes anycompound and/or substance that comprises a polymer of nucleotides. Eachnucleotide is composed of a base, specifically a purine- or pyrimidinebase (i.e. cytosine (C), guanine (G), adenine (A), thymine (T) or uracil(U)), a sugar (i.e. deoxyribose or ribose), and a phosphate group.Often, the nucleic acid molecule is described by the sequence of bases,whereby said bases represent the primary structure (linear structure) ofa nucleic acid molecule. The sequence of bases is typically representedfrom 5′ to 3′. Herein, the term nucleic acid molecule encompassesdeoxyribonucleic acid (DNA) including e.g., complementary DNA (cDNA) andgenomic DNA, ribonucleic acid (RNA), in particular messenger RNA (mRNA),synthetic forms of DNA or RNA, and mixed polymers comprising two or moreof these molecules. The nucleic acid molecule may be linear or circular.In addition, the term nucleic acid molecule includes both, sense andantisense strands, as well as single stranded and double stranded forms.Moreover, the herein described nucleic acid molecule can containnaturally occurring or non-naturally occurring nucleotides. Examples ofnon-naturally occurring nucleotides include modified nucleotide baseswith derivatized sugars or phosphate backbone linkages or chemicallymodified residues. Nucleic acid molecules also encompass DNA and RNAmolecules which are suitable as a vector for direct expression of anantibody of the invention in vitro and/or in vivo, e.g., in a host orpatient. Such DNA (e.g., cDNA) or RNA (e.g., mRNA) vectors, can beunmodified or modified. For example, mRNA can be chemically modified toenhance the stability of the RNA vector and/or expression of the encodedmolecule so that mRNA can be injected into a subject to generate theantibody in vivo (see e.g., Stadler et al, Nature Medicine 2017,published online 12 Jun. 2017, doi:10.1038/nm.4356 or EP 2 101 823 B1).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. In certain embodiments, vectors direct theexpression of nucleic acids to which they are operatively linked. Suchvectors are referred to herein as “expression vectors.”

As used herein, the term “homologous sequences” refers to sequences thatshare a significant sequence similarity as determined by an alignment ofthe sequences. For example, two sequences can be about 50%, 60%, 70%,80%, 90%, 95%, 99%, or 99.9% homologous. The alignment is carried out byalgorithms and computer programs including, but not limited to, BLAST,FASTA, and HMME, which compares sequences and calculates the statisticalsignificance of matches based on factors such as sequence length,sequence identify and similarity, and the presence and length ofsequence mismatches and gaps. Homologous sequences can refer to both DNAand protein sequences.

As used herein, the term “flanking” refers to that a first nucleotidesequence is located at either a 5′ or 3′ end, or both ends of a secondnucleotide sequence. The flanking nucleotide sequence can be adjacent toor at a defined distance from the second nucleotide sequence. There isno specific limit of the length of a flanking nucleotide sequence. Forexample, a flanking sequence can be a few base pairs or a few thousandbase pairs. In certain embodiments, the length of a flanking nucleotidesequence can be about at least 15 base pairs, at least 20 base pairs, atleast 30 base pairs, at least 40 base pairs, at least 50 base pairs, atleast 75 base pairs, at least 100 base pairs, at least 150 base pairs,at least 200 base pairs, at least 300 base pairs, at least 400 basepairs, at least 500 base pairs, at least 1,000 base pairs, at least1,500 base pairs, at least 2,000 base pairs, at least 3,000 base pairs,at least 4,000 base pairs, at least 5,000 base pairs, at least 6,000base pairs, at least 7,000 base pairs, at least 8,000 base pairs, atleast 9,000 base pairs, at least 10,000 base pairs.

As used herein, the term “exogenous” indicates that a nucleotidesequence does not originate from a host cell and is introduced into ahost cell by traditional DNA delivery methods, e.g., by transfection,electroporation, or transformation methods. The term “endogenous” refersto that a nucleotide sequence originates from a host cell. An“exogenous” nucleotide sequence can have an “endogenous” counterpartthat is identical in base compositions, but where the “exogenous”sequence is introduced into the host cell, e.g., via recombinant DNAtechnology.

2. Integration Sites

The presently disclosed subject matter provides a host cell suitable fortargeted integration of exogenous nucleotide sequences. In certainembodiments, the host cell comprises an exogenous nucleotide sequenceintegrated at an integration site on the genome of the host cell, i.e.,a TI host cell.

An “integration site” comprises a nucleic acid sequence within a hostcell genome into which an exogenous nucleotide sequence is inserted. Incertain embodiments, an integration site is between two adjacentnucleotides on the host cell genome. In certain embodiments, anintegration site includes a stretch of nucleotides between any of whichan exogenous nucleotide sequence can be inserted. In certainembodiments, the integration site is located within a specific locus ofthe genome of the TI host cell. In certain embodiments, the integrationsite is within an endogenous gene of the TI host cell.

In certain embodiments, the exogenous nucleotide sequence is integratedat a site within a specific locus of the genome of a TI host cell. Incertain embodiments, the locus into which the exogenous nucleotidesequence is integrated is at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, at least about 99%, or at least about 99.9% homologous to asequence selected from SEQ ID Nos. 1-7.

In certain embodiments, the exogenous nucleotide sequence is integratedat a site within a specific locus of the genome of a TI host cell. Incertain embodiments, the locus into which the exogenous nucleotidesequence is integrated is at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, at least about 99%, or at least about 99.9% homologous to asequence selected from Contigs NW_006874047.1, NW_006884592.1,NW_006881296.1, NW_003616412.1, NW_003615063.1, NW_006882936.1, andNW_003615411.1.

In certain embodiments, the exogenous nucleotide sequence is integratedat an integration site located within a position selected fromnucleotides numbered 1-1,000 bp; 1,000-2,000 bp; 2,000-3,000 bp;3,000-4,000 bp; and 4,000-4,301 bp of SEQ ID No. 1. In certainembodiments, the exogenous nucleotide sequence is integrated at anintegration site located within a position selected from nucleotidesnumbered 1-100,000 bp; 100,000-200,000 bp; 200,000-300,000 bp;300,000-400,000 bp; 400,000-500,000 bp; 500,000-600,000 bp;600,000-700,000 bp; and 700,000-728785 bp of SEQ ID No. 2. In certainembodiments, the exogenous nucleotide sequence is integrated at anintegration site located within a position selected from nucleotidesnumbered 1-100,000 bp; 100,000-200,000 bp; 200,000-300,000 bp;300,000-400,000 bp; and 400,000-413,983 of SEQ ID No. 3. In certainembodiments, the exogenous nucleotide sequence is integrated at anintegration site located within a position selected from nucleotidesnumbered 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; and30,000-30,757 bp of SEQ ID No. 4. In certain embodiments, the exogenousnucleotide sequence is integrated at an integration site located withina position selected from nucleotides numbered 1-10,000 bp; 10,000-20,000bp; 20,000-30,000 bp; 30,000-40,000 bp; 40,000-50,000 bp; 50,000-60,000bp; and 60,000-68,962 bp of SEQ ID No. 5. In certain embodiments, theexogenous nucleotide sequence is integrated at an integration sitelocated within a position selected from nucleotides numbered 1-10,000bp; 10,000-20,000 bp; 20,000-30,000 bp; 30,000-40,000 bp; 40,000-50,000bp; and 50,000-51,326 bp of SEQ ID No. 6. In certain embodiments, theexogenous nucleotide sequence is integrated at an integration sitelocated within a position selected from nucleotides numbered 1-10,000bp; 10,000-20,000 bp; and 20,000-22,904 bp of SEQ ID No. 7.

In certain embodiments, the nucleotide sequence immediately 5′ of theintegrated exogenous sequence is selected from the group consisting ofnucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 ofNW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides69303-79768 of NW_003616412.1, nucleotides 293481-315265 ofNW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, ornucleotides 82214-97705 of NW_003615411.1 and sequences at least 50%homologous thereto. In certain embodiments, the nucleotide sequenceimmediately 5′ of the integrated exogenous sequence are at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 99%, or at leastabout 99.9% homologous to nucleotides 41190-45269 of NW_006874047.1,nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 ofNW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 ofNW_006882936.1, or nucleotides 82214-97705 of NW_003615411.1.

In certain embodiments, the nucleotide sequence immediately 3′ of theintegrated exogenous sequence is selected from the group consisting ofnucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 ofNW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides79769-100059 of NW_003616412.1, nucleotides 315266-362442 ofNW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, ornucleotides 97706-105117 of NW_003615411.1 and sequences at least 50%homologous thereto. In certain embodiments, the nucleotide sequenceimmediately 3′ of the integrated exogenous sequence is at least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 99%, or at leastabout 99.9% homologous to nucleotides 45270-45490 of NW_006874047.1,nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1,nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768of NW_006882936.1, or nucleotides 97706-105117 of NW_003615411.1.

In certain embodiments, the integrated exogenous nucleotide sequence isoperably linked to a nucleotide sequence selected from the groupconsisting of SEQ ID. Nos. 1-7 and sequences at least 50% homologousthereto. In certain embodiments, the nucleotide sequence operably linkedto the exogenous nucleotide sequence is at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, at least about 99%, or at least about 99.9%homologous to a sequence selected from SEQ ID Nos. 1-7. In certainembodiments, the integrated exogenous nucleotide sequence comprises atleast one SOI. In certain embodiments, the operably linked nucleotidesequence increases the expression level of the SOI compared to arandomly integrated SOI. In certain embodiments, the integratedexogenous SOI is expressed at about 20%, 30%, 40%, 50%, 100%, 2 fold, 3fold, 5 fold, or 10 fold higher than a randomly integrated SOI.

In certain embodiments, the integrated exogenous sequence is flanked 5′by a nucleotide sequence selected from the group consisting ofnucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 ofNW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides69303-79768 of NW_003616412.1, nucleotides 293481-315265 ofNW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, andnucleotides 82214-97705 of NW_003615411.1. and sequences at least 50%homologous thereto, and is flanked 3′ by a nucleotide sequence selectedfrom the group consisting of nucleotides 45270-45490 of NW_006874047.1,nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1,nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768of NW_006882936.1, and nucleotides 97706-105117 of NW_003615411.1 andsequences at least 50% homologous thereto. In certain embodiments, thenucleotide sequence flanking 5′ of the integrated exogenous nucleotidesequence is at least about 50%, at least about 60%, at least about 70%,at least about 80%, at least about 90%, at least about 95%, at leastabout 99%, or at least about 99.9% homologous to nucleotides 41190-45269of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1,nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 ofNW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 ofNW_003615411.1, and the nucleotide sequences flanking 3′ of theintegrated exogenous nucleotide sequence is at least about 50%, at leastabout 60%, at least about 70%, at least about 80%, at least about 90%,at least about 95%, at least about 99%, or at least about 99.9%homologous to SEQ ID Nos. nucleotides 45270-45490 of NW_006874047.1,nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1,nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768of NW_006882936.1, and nucleotides 97706-105117 of NW_003615411.1.

In certain embodiments, the integrated exogenous nucleotide isintegrated into a locus immediately adjacent to all or a portion of asequence selected from the group consisting of sequences at least about90% homologous to a sequence selected from SEQ ID Nos. 1-7.

In certain embodiments, the integrated exogenous nucleotide sequence isadjacent to a nucleotide sequence selected from the group consisting ofSEQ ID. Nos. 1-7 and sequences at least 50% homologous thereto. Incertain embodiments, the integrated exogenous nucleotide sequence iswithin about 100 bp, about 200 bp, about 500 bp, about 1 kb distancefrom a sequence selected from the group consisting of SEQ ID. Nos. 1-7and sequences at least 50% homologous thereto. In certain embodiments,the nucleotide sequence adjacent to the exogenous nucleotide sequence isat least about 50%, at least about 60%, at least about 70%, at leastabout 80%, at least about 90%, at least about 95%, at least about 99%,or at least about 99.9% homologous to a sequence selected from SEQ IDNos. 1-7.

In certain embodiments, the exogenous nucleotide sequence is integratedat an integration site adjacent to a position selected from nucleotidesnumbered 1-1,000 bp; 1,000-2,000 bp; 2,000-3,000 bp; 3,000-4,000 bp; and4,000-4,301 bp of SEQ ID No. 1. In certain embodiments, the exogenousnucleotide sequence is integrated at an integration site adjacent to aposition selected from nucleotides numbered 1-100,000 bp;100,000-200,000 bp; 200,000-300,000 bp; 300,000-400,000 bp;400,000-500,000 bp; 500,000-600,000 bp; 600,000-700,000 bp; and700,000-728785 bp of SEQ ID No. 2. In certain embodiments, the exogenousnucleotide sequence is integrated at an integration site adjacent to aposition selected from nucleotides numbered 1-100,000 bp;100,000-200,000 bp; 200,000-300,000 bp; 300,000-400,000 bp; and400,000-413,983 of SEQ ID No. 3. In certain embodiments, the exogenousnucleotide sequence is integrated at an integration site adjacent to aposition selected from nucleotides numbered 1-10,000 bp; 10,000-20,000bp; 20,000-30,000 bp; and 30,000-30,757 bp of SEQ ID No. 4. In certainembodiments, the exogenous nucleotide sequence is integrated at anintegration site adjacent to a position selected from nucleotidesnumbered 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp; 30,000-40,000bp; 40,000-50,000 bp; 50,000-60,000 bp; and 60,000-68,962 bp of SEQ IDNo. 5. In certain embodiments, the exogenous nucleotide sequence isintegrated at an integration site adjacent to a position selected fromnucleotides numbered 1-10,000 bp; 10,000-20,000 bp; 20,000-30,000 bp;30,000-40,000 bp; 40,000-50,000 bp; and 50,000-51,326 bp of SEQ ID No.6. In certain embodiments, the exogenous nucleotide sequence isintegrated at an integration site adjacent to a position selected fromnucleotides numbered 1-10,000 bp; 10,000-20,000 bp; and 20,000-22,904 bpof SEQ ID No. 7.

In certain embodiments, the locus comprising the integration site of theexogenous nucleotide sequence does not encode an open reading frame(ORF). In certain embodiments, the locus comprising the integration siteof the exogenous nucleotide sequence includes cis-acting elements, e.g.,promoters and enhancers. In certain embodiments, the locus comprisingthe integration site of the exogenous nucleotide sequence is free of anycis-acting elements, e.g., promoters and enhancers, that enhance geneexpression.

In certain embodiments, an exogenous nucleotide sequence is integratedat an integration site within an endogenous gene selected from the groupconsisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2,MTMR2, and XP_003512331.2. The endogenous LOC107977062, LOC100768845,ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes include thewild-type and all homologous sequences of LOC107977062, LOC100768845,ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes. In certainembodiments, the homologous sequences of LOC107977062, LOC100768845,ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes can be atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 99%, or atleast about 99.9% homologous to the wild-type LOC107977062,LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes.In certain embodiments, the LOC107977062, LOC100768845, ITPR2,ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes are wild-typemammalian LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2,and XP_003512331.2 genes. In certain embodiments, the LOC107977062,LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genesare wild-type human LOC107977062, LOC100768845, ITPR2, ERE67000.1,UBAP2, MTMR2, and XP_003512331.2 genes. In certain embodiments, theLOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, andXP_003512331.2 genes are wild-type hamster LOC107977062, LOC100768845,ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 genes.

In certain embodiments, the integration site is operably linked to anendogenous gene selected from the group consisting of LOC107977062,LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and atleast about 90% homologous sequences thereof. In certain embodiments,the integration site is flanked by an endogenous gene selected from thegroup consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1,UBAP2, MTMR2, XP_003512331.2, and at least about 90% homologoussequences thereof.

Table 1 provides exemplary TI host cell integration sites:

TABLE 1 TI host cell integration sites Contig Integration Host ContigSize (kb) site (bp) Gene (SEQ ID No.) 1 NW_006874047.1 727 45269LOC107977062 (SEQ ID No. 1) 2 NW_006884592.1 931 207911 LOC100768845(SEQ ID No. 2) 3 NW_006881296.1 1016 491909 ITPR2 (SEQ ID No. 3) 4NW_003616412.1 127 79768 ERE67000.1 (SEQ ID No. 4) 5 NW_003615063.1 372315265 UBAP2 (SEQ ID No. 5) 6 NW_006882936.1 3042 2662054 MTMR2 (SEQ IDNo. 6) 7 NW_003615411.1 277 97706 XP_003512331.2 (SEQ ID No. 7)

In certain embodiments, an integration site and/or the nucleotidesequences flanking the integration site can be identifiedexperimentally. In certain embodiments, an integration site and/or thenucleotide sequences flanking the integration site can be identified bygenome-wide screening approaches to isolate host cells that express, ata desirable level, a polypeptide of interest encoded by one or more SOIsintegrated into one or more exogenous nucleotide sequences, where theexogenous sequences are themselves integrated into one or more loci inthe genome of the host cell. In certain embodiments, an integration siteand/or the nucleotide sequences flanking an integration site can beidentified by genome-wide screening approaches followingtransposase-based cassette integration event. In certain embodiments, anintegration site and/or the nucleotide sequences flanking an integrationsite can be identified by brute force random integration screening. Incertain embodiments, an integration site and/or the nucleotide sequencesflanking an integration site can be determined by conventionalsequencing approaches such as target locus amplification (TLA) followedby next-generation sequencing (NGS) and whole-genome NGS. In certainembodiments, the location of an integration site on a chromosome can bedetermined by conventional cell biology approaches such as fluorescencein-situ hybridization (FISH) analysis.

In certain embodiments, a TI host cell comprises a first exogenousnucleotide sequence integrated at a first integration site within aspecific first locus in the genome of the TI host cell and a secondexogenous nucleotide sequence integrated at a second integration sitewithin a specific second locus in the genome. In certain embodiments, aTI host cell comprises multiple exogenous nucleotide sequencesintegrated at multiple integration sites in the genome of the TI hostcell.

In certain embodiments, the TI host cells of the present disclosurecomprise at least two distinct exogenous nucleotide sequences, e.g.,exogenous nucleotide sequences comprising at least one RRS. In certainembodiments, the two or more exogenous nucleotide sequences can betargeted for the introduction of one or more SOIs. In certainembodiments the SOIs are the same. In certain embodiments, the SOIs aredistinct. In certain embodiments, a parental TI host cell comprising afirst exogenous nucleotide sequence can comprise a second exogenousnucleotide sequence at an integration site that is different from theintegration site of the first exogenous nucleotide sequence.

In certain embodiments, the integration site is at least about 50%, atleast about 60%, at least about 70%, at least about 80%, at least about90%, at least about 95%, at least about 99%, or at least about 99.9%homologous to a sequence selected from SEQ ID Nos. 1-7. In certainembodiments, the integration sites can be on the same chromosome. Incertain embodiments, the integration sites are located within 1-1,000nucleotides, 1,000-100,000 nucleotides, 100,000-1,000,000 nucleotides ormore from each other in the same chromosome. In certain embodiments theintegration sites are on different chromosomes. In certain embodiments,a TI host cell comprising an exogenous nucleotide sequence at oneintegration site can be used for the insertion of at least two, at leastthree, at least four, at least five, at least six, at least 7, at least8, or more exogenous nucleotide sequences at the same or differentintegration sites.

In certain embodiments, the feasibility of recombinase-mediated cassetteexchange (RMCE) of at least two integration sites can be evaluated foreach site individually. In certain embodiments, the feasibility of RMCEat least two integration sites can be evaluated simultaneously. Thefeasibility of RMCE at multiple sites can evaluated by methods known inthe art, e.g., measuring the polypeptide titer, or the polypeptidespecific production. In certain embodiments, the evaluation can beperformed by methods known in the art, e.g., by evaluating the titerand/or specific productivity of a culture of the TI host cell expressingthe SOI(s). Exemplary culture strategies include, but are not limitedto, fed-batch shake flask cultures and a bioreactor fed-batch cultures.Titer and specific productivity of the TI host cells expressing apolypeptide of interest can evaluated by methods known in the art, e.g.,but not limited to, ELISA, FACS, Fluorometric Microvolume AssayTechnology (FMAT), protein-A affinity chromatography, Western blotanalysis.

3. Exogenous Nucleotide Sequences

An exogenous nucleotide sequence is a nucleotide sequence that does notoriginate from a host cell but can be introduced into a host cell bytraditional DNA delivery methods, e.g., by transfection,electroporation, or transformation methods. In certain embodiments, theexogenous nucleotide sequence is a sequence of interest (SOI), e.g., anucleotide sequence encoding a polypeptide of interest. In certainembodiments, however, the exogenous nucleotide sequences employed in thecontext of the instant disclosure comprises elements, e.g., one or morerecombination recognition sequences (RRs) and one or more selectionmarkers, which facilitate the introduction of additional nucleic acidsequences, e.g., SOIs. In certain embodiments, the exogenous nucleotidesequences facilitating the introduction of additional nucleic acidsequences are referred to herein as “landing pads.” Accordingly, incertain embodiments, a TI host cell can comprise: (1) an exogenousnucleotide sequence that includes one or more SOIs, e.g., an SOIincorporated into a particular locus in a host cell genome via anexogenous site-specific nuclease mediated (e.g., CRISPR/Cas9-mediated)targeted integration; (2) an exogenous nucleotide sequence that includesone or more landing pads; or (3) an exogenous nucleotide sequence thatincludes one or more landing pads into which one or more SOIs have beenincorporated.

In certain embodiments, a TI host cell comprises at least one exogenousnucleotide sequence integrated at one or more integration sites in thegenome of the TI host cell. In certain embodiments, the exogenousnucleotide sequence is integrated at one or more integration siteswithin a specific a locus of the genome of the TI host cell. Forexample, but not by way of limitation, at least one exogenous nucleicacid sequence can be integrated at one or more locus having least about50%, at least about 60%, at least about 70%, at least about 80%, atleast about 90%, at least about 95%, at least about 99%, or at leastabout 99.9% homologous to a sequence selected from SEQ ID Nos. 1-7.

3.1 Landing Pads

In certain embodiments, an integrated exogenous nucleotide sequencecomprises one or more recombination recognition sequence (RRS), whereinthe RRS can be recognized by a recombinase. In certain embodiments, theintegrated exogenous nucleotide sequence comprises at least two RRSs. Incertain embodiments, the integrated exogenous nucleotide sequencecomprises two RRSs and the two RRSs are the same. In certainembodiments, the integrated exogenous nucleotide sequence comprises twoRRSs and the two RRSs are heterospecific, i.e., not recognized by thesame recombinase. In certain embodiments, an integrated exogenousnucleotide sequence comprises three RRSs, wherein the third RRS islocated between the first and the second RRS. In certain embodiments,the first and the second RRS are the same and the third RRS is differentfrom the first or the second RRS. In certain embodiments, all three RRSsare heterospecific. In certain embodiments, an integrated exogenousnucleotide sequence comprises four, five, six, seven, or eight RRSs. Incertain embodiments, an integrated exogenous nucleotide sequencecomprises multiple RRSs. In certain embodiments, the multiple two ormore RRSs are the same. In certain embodiments, the two or more RRSs areheterospecific. In certain embodiments each RRS can be recognized by adistinct recombinase. In certain embodiments, the subset of the totalnumber of RRSs are the homospecific, i.e., recognized by the samerecombinase, and a subset of the total number of RRSs areheterospecific, i.e., not recognized by the same recombinase. In certainembodiments, the RRS or RRSs can be selected from the group consistingof a LoxP sequence, a LoxP L3 sequence, a LoxP 2 L sequence, a LoxFassequence, a Lox511 sequence, a Lox2272 sequence, a Lox2372 sequence, aLox5171 sequence, a Loxm2 sequence, a Lox71 sequence, a Lox66 sequence,a FRT sequence, a Bxb1 attP sequence, a Bxb1 attB sequence, a pC31 attPsequence, and a pC31 attB sequence.

In certain embodiments, the integrated exogenous nucleotide sequencecomprises at least one selection marker. In certain embodiments, theintegrated exogenous nucleotide sequence comprises one RRS and at leastone selection marker. In certain embodiments, the integrated exogenousnucleotide sequence comprises a first and a second RRS, and at least oneselection marker. In certain embodiments, a selection marker is locatedbetween the first and the second RRS. In certain embodiments, two RRSsflank at least one selection marker, i.e., a first RRS is located 5′upstream and a second RRS is located 3′ downstream of the selectionmarker. In certain embodiments, a first RRS is adjacent to the 5′ end ofthe selection marker and a second RRS is adjacent to the 3′ end of theselection marker.

In certain embodiments, a selection marker is located between a firstand a second RRS and the two flanking RRSs are the same. In certainembodiments, the two RRSs flanking the selection marker are both LoxPsequences. In certain embodiments, the two RRSs flanking the selectionmarker are both FRT sequences. In certain embodiments, a selectionmarker is located between a first and a second RRS and the two flankingRRSs are heterospecific. In certain embodiments, the first flanking RRSis a LoxP L3 sequence and the second flanking RRS is a LoxP 2 Lsequence. In certain embodiments, a LoxP L3 sequenced is located 5′ ofthe selection marker and a LoxP 2 L sequence is located 3′ of theselection marker. In certain embodiments, the first flanking RRS is awild-type FRT sequence and the second flanking RRS is a mutant FRTsequence. In certain embodiments, the first flanking RRS is a Bxb1 attPsequence and the second flanking RRS is a Bxb1 attB sequence. In certainembodiments, the first flanking RRS is a φC31 attP sequence and thesecond flanking RRS is a φC31 attB sequence. In certain embodiments, thetwo RRSs are positioned in the same orientation. In certain embodiments,the two RRSs are both in the forward or reverse orientation. In certainembodiments, the two RRSs are positioned in opposite orientation.

In certain embodiments, a selection marker can be an aminoglycosidephosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG),neomycin and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase(TK), glutamine synthetase (GS), asparagine synthetase, tryptophansynthetase (indole), histidinol dehydrogenase (histidinol D), and genesencoding resistance to puromycin, blasticidin, bleomycin, phleomycin,chloramphenicol, Zeocin, or mycophenolic acid. In certain embodiments, aselection marker can be a GFP, an eGFP, a synthetic GFP, a YFP, an eYFP,a CFP, an mPlum, an mCherry, a tdTomato, an mStrawberry, a J-red, aDsRed-monomer, an mOrange, an mKO, an mCitrine, a Venus, a YPet, anEmerald, a CyPet, an mCFPm, a Cerulean, or a T-Sapphire marker. Incertain embodiments, the selection marker can be a fusion constructcomprising at least two selection markers. In certain embodiments thegene encoding a selection marker or a fragment of the selection markercan be fused to the gene encoding a different selection marker or afragment thereof.

In certain embodiments, the integrated exogenous nucleotide sequencecomprises two selection markers flanked by two RRSs, wherein a firstselection marker is different from a second selection marker. In certainembodiments, the two selection markers are both selected from the groupconsisting of a glutamine synthetase selection marker, a thymidinekinase selection marker, a HYG selection marker, and a puromycinresistance selection marker. In certain embodiments, the integratedexogenous nucleotide sequence comprises a thymidine kinase selectionmarker and a HYG selection marker. In certain embodiments, the firstselection maker is selected from the group consisting of anaminoglycoside phosphotransferase (APH) (e.g., hygromycinphosphotransferase (HYG), neomycin and G418 APH), dihydrofolatereductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS),asparagine synthetase, tryptophan synthetase (indole), histidinoldehydrogenase (histidinol D), and genes encoding resistance topuromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, Zeocin,and mycophenolic acid, and the second selection maker is selected fromthe group consisting of a GFP, an eGFP, a synthetic GFP, a YFP, an eYFP,a CFP, an mPlum, an mCherry, a tdTomato, an mStrawberry, a J-red, aDsRed-monomer, an mOrange, an mKO, an mCitrine, a Venus, a YPet, anEmerald, a CyPet, an mCFPm, a Cerulean, and a T-Sapphire marker. Incertain embodiments, the first selection marker is a glutaminesynthetase selection marker and the second selection marker is a GFPmarker. In certain embodiments, the two RRSs flanking both selectionmarkers are the same. In certain embodiments, the two RRSs flanking bothselection markers are different.

In certain embodiments, the selection marker is operably linked to apromoter sequence. In certain embodiments, the selection marker isoperably linked to an SV40 promoter. In certain embodiments, theselection marker is operably linked to a Cytomegalovirus (CMV) promoter.

In certain embodiments, the integrated exogenous nucleotide sequencecomprises at least one selection marker and an IRES, wherein the RES isoperably linked to the selection marker. In certain embodiments, theselection marker operably linked to the IRES is selected from the groupconsisting of a GFP, an eGFP, a synthetic GFP, a YFP, an eYFP, a CFP, anmPlum, an mCherry, a tdTomato, an mStrawberry, a J-red, a DsRed-monomer,an mOrange, an mKO, an mCitrine, a Venus, a YPet, an Emerald, a CyPet,an mCFPm, a Cerulean, and a T-Sapphire marker. In certain embodiments,the selection marker operably linked to the IRES is a GFP marker. Incertain embodiments, the integrated exogenous nucleotide sequencecomprises an IRES and two selection markers flanked by two RRSs, whereinthe IRES is operably linked to the second selection marker. In certainembodiments, the integrated exogenous nucleotide sequence comprises anIRES and three selection markers flanked by two RRSs, wherein the RES isoperably linked to the third selection marker. In certain embodiments,the integrated exogenous nucleotide sequence comprises an IRES and threeselection markers flanked by two RRSs, wherein the IRES is operablylinked to the third selection marker. In certain embodiments, the thirdselection marker is different from the first or the second selectionmarker. In certain embodiments, the integrated exogenous nucleotidesequence comprises a first selection marker operably linked to apromoter and a second selection marker operably linked to an TRES. Incertain embodiments, the integrated exogenous nucleotide sequencecomprises a glutamine synthetase selection marker operably linked to aSV40 promoter and a GFP selection marker operably linked to an RES. Incertain embodiments, the integrated exogenous nucleotide sequencecomprises a thymidine kinase selection marker and a HYG selection markeroperably linked to a CMV promoter and a GFP selection marker operablylinked to an IRES.

In certain embodiments, the integrated exogenous nucleotide sequencecomprises three RRSs. In certain embodiments, the third RRS is locatedbetween the first and the second RRS. In certain embodiments, all threeRRSs are the same. In certain embodiments, the first and the second RRSare the same, and the third RRS is different from the first or thesecond RRS. In certain embodiments, all three RRSs are heterospecific.

3.2 Sequences of Interest (SOIs)

In certain embodiments, the integrated exogenous nucleotide sequencecomprises at least one exogenous SOI. In certain embodiments, theintegrated exogenous nucleotide sequence comprises at least oneselection marker and at least one exogenous SOI. In certain embodiments,the integrated exogenous nucleotide sequence comprises at least oneselection marker, at least one exogenous SOI, and at least one RRS. Incertain embodiments, the integrated exogenous nucleotide sequencecomprises at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight or more SOIs.In certain embodiments the SOIs are the same. In certain embodiments,the SOIs are different.

In certain embodiments the SOI encodes a single chain antibody orfragment thereof. In certain embodiments, the SOI encodes an antibodyheavy chain sequence or fragment thereof. In certain embodiments, theSOI encodes an antibody light chain sequence or fragment thereof. Incertain embodiments, an integrated exogenous nucleotide sequencecomprises an SOI encoding an antibody heavy chain sequence or fragmentthereof and an SOI encoding an antibody light chain sequence or fragmentthereof. In certain embodiments, an integrated exogenous nucleotidesequence comprises an SOI encoding a first antibody heavy chain sequenceor fragment thereof, an SOI encoding a second antibody heavy chainsequence or fragment thereof, and an SOI encoding an antibody lightchain sequence or fragment thereof. In certain embodiments, anintegrated exogenous nucleotide sequence comprises an SOI encoding afirst antibody heavy chain sequence or fragment thereof, an SOI encodinga second antibody heavy chain sequence or fragment thereof, an SOIencoding a first antibody light chain sequence or fragment thereof and asecond SOI encoding an antibody light chain sequence or fragmentthereof. In certain embodiments, the number of SOIs encoding for heavyand light chain sequences can be selected to achieve a desiredexpression level of the heavy and light chain polypeptides, e.g., toachieve a desired amount of bispecific antibody production. In certainembodiments, the individual SOIs encoding heavy and light chainsequences can be integrated, e.g., into a single exogenous nucleic acidsequence present at a single integration site, into multiple exogenousnucleic acid sequences present at a single integration site, or intomultiple exogenous nucleic acid sequences integrated at distinctintegration sites within the TI host cell.

In certain embodiments, the integrated exogenous nucleotide sequencecomprises at least one selection marker, at least one exogenous SOI, andone RRS. In certain embodiments, the RRS is located adjacent to at leastone selection marker or at least one exogenous SOI. In certainembodiments, the integrated exogenous nucleotide sequence comprises atleast one selection marker, at least one exogenous SOI, and two RRSs. Incertain embodiments, the integrated exogenous nucleotide sequencecomprises at least one selection marker and at least one exogenous SOIlocated between the first and the second RRS. In certain embodiments,the two RRSs flanking the selection marker and the exogenous SOI are thesame. In certain embodiments, the two RRSs flanking the selection markerand the exogenous SOI are different. In certain embodiments, the firstflanking RRS is a LoxP L3 sequence and the second flanking RRS is a LoxP2 L sequence. In certain embodiments, a L3 LoxP sequenced is located 5′of the selection marker and the exogenous SOI, and a LoxP 2 L sequenceis located 3′ of the selection marker and the exogenous SOI.

In certain embodiments, the integrated exogenous nucleotide sequencecomprises three RRSs and two exogenous SOIs, and the third RRS islocated between the first and the second RRS. In certain embodiments,the first SOI is located between the first and the third RRS, and thesecond SOI is located between the third and the second RRS. In certainembodiments, the first and the second SOI are different. In certainembodiments, the first and the second RRS are the same and the third RRSis different from the first or the second RRS. In certain embodiments,all three RRSs are heterospecific. In certain embodiments, the first RRSis a LoxP L3 site, the second RRS is a LoxP 2 L site, and the third RRSis a LoxFas site. In certain embodiments, the integrated exogenousnucleotide sequence comprises three RRSs, one exogenous SOI, and oneselection marker. In certain embodiments, the SOI is located between thefirst and the third RRS, and the selection marker is located between thethird and the second RRS. In certain embodiments, the integratedexogenous nucleotide sequence comprises three RRSs, two exogenous SOIs,and one selection marker. In certain embodiments, the first SOI and theselection marker are located between the first and the third RRS, andthe second SOI is located between the third and the second RRS.

In certain embodiments, the exogenous SOI encodes a polypeptide ofinterest. Such polypeptides of interest can be selected from the groupincluding, but not limited to, an antibody, an enzyme, a cytokine, agrowth factor, a hormone, a viral protein, a bacterial protein, avaccine protein, or a protein with therapeutic function. In certainembodiments, the exogenous SOI encodes an antibody or an antigen-bindingfragment thereof. In certain embodiments, the exogenous SOI encodes asingle chain antibody, an antibody light chain, an antibody heavy chain,a single-chain Fv fragment (scFv), or an Fc fusion protein. In certainembodiments, the exogenous SOI (or SOIs) encodes a standard antibody. Incertain embodiments, the exogenous SOI (or SOIs) encodes ahalf-antibody, for example, but not limited to, antibodies B, Q, T andmAb I of the present disclosure. In certain embodiments, the exogenousSOI (or SOIs) encodes a complex antibody. In certain embodiments, thecomplex antibody can be a bispecific antibody, for example, but notlimited to, Bispecific Molecule A, Bispecific Molecule B, BispecificMolecule C, or Bispecific Molecule D of the present disclosure. Incertain embodiments, the exogenous SOI is operably linked to at leastone cis-acting element, for example, a promoter or an enhancer. Incertain embodiments, the exogenous SOI is operably linked to a CMVpromoter.

In certain embodiments, the integrated exogenous nucleotide sequencecomprises two RRSs and at least two exogenous SOIs located between thetwo RRSs. In certain embodiments, SOIs encoding one heavy chain and onelight chain of an antibody are located between the two RRSs. In certainembodiments, SOIs encoding one heavy chain and two light chains of anantibody are located between the two RRSs. In certain embodiments, SOIsencoding different combinations of copies of heavy chain and light chainof an antibody are located between the two RRSs.

In certain embodiments, the integrated exogenous nucleotide sequencecomprises three RRSs and at least two exogenous SOIs, and the third RRSis located between the first and the second RRS. In certain embodiments,at least one SOI is located between the first and the third RRS, and atleast one SOI is located between the third and the second RRS. Incertain embodiments, the first and the second RRS are the same and thethird RRS is different from the first or the second RRS. In certainembodiments, all three RRSs are heterospecific. In certain embodiments,SOIs encoding one heavy chain and one light chain of a first antibodyare located between the first and the third RRS, and SOIs encoding oneheavy chain and one light chain of a second antibody are located betweenthe third and the second RRS. In certain embodiments, SOIs encoding oneheavy chain and two light chains of a first antibody are located betweenthe first and the third RRS, and SOIs encoding one heavy chain and onelight chain of a second antibody are located between the third RRS andthe second RRS. In certain embodiments, SOIs encoding one heavy chainand three light chains of a first antibody are located between the firstand the third RRS, and SOIs encoding one light chain of the firstantibody and one heavy chain and one light chain of a second antibodyare located between the third RRS and the second RRS. In certainembodiments, SOIs encoding one heavy chain and three light chains of afirst antibody are located between the first and the third RRS, and SOIsencoding two light chains of the first antibody and one heavy chain andone light chain of a second antibody are located between the third RRSand the second RRS. In certain embodiments, SOIs encoding differentcombinations of copies of heavy chains and light chains of multipleantibodies are located between the first and the third RRS, and betweenthe third and the second RRS.

In certain embodiments, the number of SOIs is selected to increase thetiter and/or specific productivity of the host cells expressing theSOIs. For example, but not by way of limitation, the incorporation oftwo, three, four, five, six, seven, eight, or more SOIs can result inincreased titer and/or specific productivity.

In the context of antibody expression, the inclusion of an additionalheavy or light chain encoding SOIs can result in increased titer and/orspecific productivity. For example, but not by way of limitation, whenincreasing copy number from one heavy chain and one light chain (HL) toone heavy chain and two light chain encoding sequences (HLL), anincrease in titer and/or specific productivity can be achieved.Similarly, as outlined in the examples below, increasing from HLL (threeSOIs) to HLL-HL (five SOIs) or HLL-HLL (six SOIs) can provide for anincrease in titer and/or specific productivity. Additionally, increasingcopy number to HLL-HL (five SOIs) or HLL-HLHL (seven SOIs) can providefor an increase in titer and/or specific productivity. Additionaloptions for heavy and light chain SOI copy numbers include, but are notlimited to HHL; HHL-H; HLL-H; HHL-HH; HHL-HL; HHL-LL; HLL-HH; HLL-HL;HLL-LL; HHL-HHL; HHL-HHH; HHL-HLL; HHK-LLL; HLL-HHL; HLL-HHH; HLL-LLL;HHL-HHHUL; HHL-HHHH; HHL-HHLL; HHL-HLLL; HHL-LLLL; HLL-HHHL; HLL-HHHH;HLL-HLLL; and HLL-LLLL. In certain embodiments, the inclusion ofadditional copies occurs at a single genomic locus, while in certainembodiments the SOI copies can be integrated at two or more loci, e.g.,multiple copies can be integrated at a single locus and one or morecopies integrated at one or more additional loci.

In certain embodiments, the position of the SOIs, e.g., whether one SOIis located 3′ or 5′ relative to another SOI, is selected to increase thetiter and/or specific productivity of the host cells expressing theSOIs. For example, but not by way of limitation, in the context ofantibody production, the integrated position of heavy and light chainSOIs can result in increased titer and/or specific productivity. Asoutlined in FIGS. 23A-D and the examples presented below, the relativeposition of heavy and light chain SOIs can impact the titer and specificproductivity, despite no change in SOI copy number.

4. Host Cells

The presently disclosed subject matter provides a host cell suitable fortargeted integration of nucleotide sequences and expression ofpolypeptides of interest. In certain embodiments, a host cell comprisesan endogenous gene selected from the group consisting of LOC107977062,LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2 andsequences at least 50% homologous thereto, or a locus of the genome ofthe host cell, wherein the locus comprises a nucleotide sequence that isselected from the group consisting of SEQ ID Nos. 1-7 and sequences atleast 50% homologous thereto.

In certain embodiments, a host cell is a eukaryotic host cell. Incertain embodiments, a host cell is a mammalian host cell. In certainembodiments, a host cell is a hamster host cell, a human host cell, arat host cell, or a mouse host cell. In certain embodiments, a host cellis a Chinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHOK1SV host cell, a DG44 host cell, a DUKXB-11 host cell, a CHOK1S hostcell, or a CHO K1M host cell.

In certain embodiments, a host cell is selected from the groupconsisting of monkey kidney CV1 line transformed by SV40 (COS-7), humanembryonic kidney line (293 or 293 cells as described, e.g., in Graham etal., J Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK), mousesertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod.23:243-251 (1980)), monkey kidney cells (CV1), African green monkeykidney cells (VERO-76), human cervical carcinoma cells (HELA), caninekidney cells (MDCK; buffalo rat liver cells (BRL 3A), human lung cells(W138), human liver cells (Hep G2), mouse mammary tumor (MMT 060562),TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982), MRC 5 cells, FS4 cells, YO cells, NSO cells, Sp2/0cells, and PER.C6® cells.

In certain embodiments, a host cell is a cell line. In certainembodiments, a host cell is a cell line that has been cultured for acertain number of generations. In certain embodiments, a host cell is aprimary cell.

In certain embodiments, expression of a polypeptide of interest isstable if the expression level is maintained at certain levels,increases, or decreases less than 20%, over 10, 20, 30, 50, 100, 200, or300 generations. In certain embodiments, expression of a polypeptide ofinterest is stable if the culture can be maintained without anyselection. In certain embodiments, expression of a polypeptide ofinterest is high if the polypeptide product of the gene of interestreaches about 1 g/L, about 2 g/L, about 3 g/L, about 4 g/L, about 5 g/L,about 10 g/L, about 12 g/L, about 14 g/L, or about 16 g/L.

The presently disclosed subject matter also relates to a method forproducing a polypeptide of interest. In certain embodiments, such methodcomprises: a) providing a host cell comprising at least one exogenousSOI and at least one selection marker flanked by two RRSs integratedwithin a locus of the genome of the host cell, wherein the locus is atleast about 90% homologous to a sequence selected from SEQ ID Nos. 1-7;and b) culturing the cell in a) under conditions suitable for expressingthe SOI and recovering a polypeptide of interest therefrom. In certainembodiments, such method comprises: a) providing a host cell comprisingat least two exogenous SOIs and at least one selection marker integratedwithin a locus of the genome of the host cell, wherein the locus is atleast about 90% homologous to a sequence selected from SEQ ID Nos. 1-7,wherein at least one exogenous SOI and one selection marker is flankedby a first and a third RRS and at least one exogenous SOI is flanked bya second and the third RRS; and b) culturing the cell in a) underconditions suitable for expressing the SOI and recovering a polypeptideof interest therefrom. In certain embodiments, such method comprises: a)providing a host cell comprising at least one exogenous SOI and at leastone selection marker flanked by two RRSs integrated within an endogenousgene selected from the group consisting of LOC107977062, LOC100768845,ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and at least about 90%homologous sequences thereof, and b) culturing the cell in a) underconditions suitable for expressing the SOI and recovering a polypeptideof interest therefrom. In certain embodiments, such method comprises: a)providing a host cell comprising at least two exogenous SOIs and atleast one selection marker integrated within an endogenous gene selectedfrom the group consisting of LOC107977062, LOC100768845, ITPR2,ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about90% homologous thereto, wherein at least one exogenous SOI and oneselection marker is flanked by a first and a third RRS and at least oneexogenous SOI is flanked by a second and the third RRS; and b) culturingthe cell in a) under conditions suitable for expressing the SOI andrecovering a polypeptide of interest therefrom.

In certain embodiments, polypeptide of interest is produced and secretedinto the cell culture medium. In certain embodiments, polypeptide ofinterest is expressed and retained within the host cell. In certainembodiments, polypeptide of interest is expressed, inserted into, andretained in the host cell membrane.

Exogenous nucleotides of interest or vectors can be introduced into ahost cell by conventional cell biology methods including, but notlimited to, transfection, transduction, electroporation, or injection.In certain embodiments, exogenous nucleotides of interest or vectors areintroduced into a host cell by chemical based transfection methodscomprising lipid-based transfection method, calcium phosphate-basedtransfection method, cationic polymer-based transfection method, ornanoparticle-based transfection. In certain embodiments, exogenousnucleotides of interest are introduced into a host cell byvirus-mediated transduction including, but not limited to, lentivirus,retrovirus, adenovirus, or adeno-associated virus-mediated transduction.In certain embodiments, exogenous nucleotides of interest or vectors areintroduced into a host cell via gene gun-mediated injection. In certainembodiments, both DNA and RNA molecules are introduced into a host cellusing methods described herein.

5. Targeted Integration

A targeted integration allows for exogenous nucleotide sequences to beintegrated into one or more pre-determined sites of a host cell genome.In certain embodiments, the targeted integration is mediated by arecombinase that recognizes one or more RRSs. In certain embodiments,the targeted integration is mediated by homologous recombination. Incertain embodiments, the targeted integration is mediated by anexogenous site-specific nuclease followed by HDR and/or NHEJ.

5.1. Targeted Integration Via Recombinase-Mediated Recombination

A “recombination recognition sequence” (RRS) is a nucleotide sequencerecognized by a recombinase and is necessary and sufficient forrecombinase-mediated recombination events. A RRS can be used to definethe position where a recombination event will occur in a nucleotidesequence.

In certain embodiments, a RRS is selected from the group consisting of aLoxP sequence, a LoxP L3 sequence, a LoxP 2 L sequence, a LoxFassequence, a Lox511 sequence, a Lox2272 sequence, a Lox2372 sequence, aLox5171 sequence, a Loxm2 sequence, a Lox71 sequence, a Lox66 sequence,a FRT sequence, a Bxb attP sequence, a Bxb1 attB sequence, a φC31 attPsequence, and a φC31 attB sequence.

In certain embodiments, a RRS can be recognized by a Cre recombinase. Incertain embodiments, a RRS can be recognized by a FLP recombinase. Incertain embodiments, a RRS can be recognized by a Bxb1 integrase. Incertain embodiments, a RRS can be recognized by a φC31 integrase.

In certain embodiments when the RRS is a LoxP site, the host cellrequires the Cre recombinase to perform the recombination. In certainembodiments when the RRS is a FRT site, the host cell requires the FLPrecombinase to perform the recombination. In certain embodiments whenthe RRS is a Bxb1 attP or a Bxb1 attB site, the host cell requires theBxb1 integrase to perform the recombination. In certain embodiments whenthe RRS is a φC31 attP or a φC31attB site, the host cell requires theφC31 integrase to perform the recombination. The recombinases can beintroduced into a host cell using an expression vector comprising codingsequences of the enzymes.

The Cre-LoxP site-specific recombination system has been widely used inmany biological experimental systems. Cre is a 38-kDa site-specific DNArecombinase that recognizes 34 bp LoxP sequences. Cre is derived frombacteriophase P1 and belongs to the tyrosine family site-specificrecombinase. Cre recombinase can mediate both intra and intermolecularrecombination between LoxP sequences. The LoxP sequence is composed ofan 8 bp nonpalindromic core region flanked by two 13 bp invertedrepeats. Cre recombinase binds to the 13 bp repeat thereby mediatingrecombination within the 8 bp core region. Cre-LoxP-mediatedrecombination occurs at a high efficiency and does not require any otherhost factors. If two LoxP sequences are placed in the same orientationon the same nucleotide sequence, Cre-mediated recombination will exciseDNA sequences located between the two LoxP sequences as a covalentlyclosed circle. If two LoxP sequences are placed in an inverted positionon the same nucleotide sequence, Cre-mediated recombination will invertthe orientation of the DNA sequences located between the two sequences.LoxP sequences can also be placed on different chromosomes to facilitaterecombination between different chromosomes. If two LoxP sequences areon two different DNA molecules and if one DNA molecule is circular,Cre-mediated recombination will result in integration of the circularDNA sequence.

In certain embodiments, a LoxP sequence is a wild-type LoxP sequence. Incertain embodiments, a LoxP sequence is a mutant LoxP sequence. MutantLoxP sequences have been developed to increase the efficiency ofCre-mediated integration or replacement. In certain embodiments, amutant LoxP sequence is selected from the group consisting of a LoxP L3sequence, a LoxP 2 L sequence, a LoxFas sequence, a Lox511 sequence, aLox2272 sequence, a Lox2372 sequence, a Lox5171 sequence, a Loxm2sequence, a Lox71 sequence, and a Lox66 sequence. For example, the Lox71sequence has 5 bp mutated in the left 13 bp repeat. The Lox66 sequencehas 5 bp mutated in the right 13 bp repeat. Both the wild-type and themutant LoxP sequences can mediate Cre-dependent recombination.

The FLP-FRT site-specific recombination system is similar to the Cre-Loxsystem. It involves the flippase (FLP) recombinase, which is derivedfrom the 2 μm plasmid of the yeast Saccharomyces cerevisiae. FLP alsobelongs to the tyrosine family site-specific recombinase. The FRTsequence is a 34 bp sequence that consists of two palindromic sequencesof 13 bp each flanking an 8 bp spacer. FLP binds to the 13 bppalindromic sequences and mediates DNA break, exchange and ligationwithin the 8 bp spacer. Similar to the Cre recombinase, the position andorientation of the two FRT sequences determine the outcome ofFLP-mediated recombination. In certain embodiments, a FRT sequence is awild-type FRT sequence. In certain embodiments, a FRT sequence is amutant FRT sequence. Both the wild-type and the mutant FRT sequences canmediate FLP-dependent recombination. In certain embodiments, a FRTsequence is fused to a responsive receptor domain sequence, such as, butnot limited to, a tamoxifen responsive receptor domain sequence.

Bxb1 and (pC31 belong to the serine recombinase family. They are bothderived from bacteriophages and are used by these bacteriophages toestablish lysogeny to facilitate site-specific integration of the phagegenome into the bacterial genome. These integrases catalyzesite-specific recombination events between short (40-60 bp) DNAsubstrates termed attP and attB sequences that are originally attachmentsites located on the phage DNA and bacterial DNA, respectively. Afterrecombination, two new sequences are formed, which are termed attL andattR sequences and each contains half sequences derived from attP andattB. Recombination can also occur between attL and attR sequences toexcise the integrated phage out of the bacterial DNA. Both integrasescan catalyze the recombination without the aid of any additional hostfactors. In the absence of any accessory factors, these integrasesmediate unidirectional recombination between attP and attB with greaterthan 80% efficiency. Because of the short DNA sequences that can berecognized by these integrases and the unidirectional recombination,these recombination systems have been developed as a complement to thewidely-used Cre-LoxP and FRT-FLP systems for genetic engineeringpurposes.

The terms “matching RRSs” and “homospecific RRSs” indicates that arecombination occurs between two RRSs. In certain embodiments, the twomatching RRSs are the same. In certain embodiments, both RRSs arewild-type LoxP sequences. In certain embodiments, both RRSs are mutantLoxP sequences. In certain embodiments, both RRSs are wild-type FRTsequences. In certain embodiments, both RRSs are mutant FRT sequences.In certain embodiments, the two matching RRSs are different sequencesbut can be recognized by the same recombinase. In certain embodiments,the first matching RRS is a Bxb1 attP sequence and the second matchingRRS is a Bxb1 attB sequence. In certain embodiments, the first matchingRRS is a pC31 attB sequence and the second matching RRS is a pC31 attBsequence.

In certain embodiments, an integrated exogenous nucleotide sequencecomprises two RRSs and a vector comprises two RRSs matching the two RRSson the integrated exogenous nucleotide sequence, i.e., the first RRS onthe integrated exogenous nucleotide sequence matches the first RRS onthe vector and the second RRS on the integrated exogenous nucleotidesequence matches the second RRS on the vector. In certain embodiments,the first RRS on the integrated exogenous nucleotide sequence and thefirst RRS on the vector are the same as the second RRS on the integratedexogenous nucleotide sequence and the second RRS on the vector. Anon-limiting example of such a “single-vector RMCE” strategy ispresented in FIG. 2A. In certain embodiments, the first RRS on theintegrated exogenous nucleotide sequence and the first RRS on the vectorare different from the second RRS on the integrated exogenous nucleotidesequence and the second RRS on the vector. In certain embodiments, thefirst RRS on the integrated exogenous nucleotide sequence and the firstRRS on the vector are both LoxP L3 sequences, and the second RRS on theintegrated exogenous nucleotide sequence and the second RRS on thevector are both LoxP 2 L sequences.

In certain embodiments, a “two-vector RMCE” strategy is employed. Forexample, but not by way of limitation, an integrated exogenousnucleotide sequence could comprise three RRSs, e.g., an arrangementwhere the third RRS (“RRS3”) is present between the first RRS (“RRS1”)and the second RRS (“RRS2”), while a first vector comprises two RRSsmatching the first and the third RRS on the integrated exogenousnucleotide sequence, and a second vector comprises two RRSs matching thethird and the second RRS on the integrated exogenous nucleotidesequence. An example of a two vector RMCE strategy is illustrated inFIG. 4. In such an example, RRS1, RRS2, and RRS3 are heterospecific,e.g., they do not cross-react with each other. In some embodiments, onevector (front) comprises the RRS1, a first SOI and a promoter followedby a start codon and RRS3 (in this order). The other vector (back)comprises the RRS3 fused to the coding sequence of a marker without thestart codon (ATG), an SOI 2 and the RRS2 (in this order). Additionalnucleotides may be inserted between the RRS3 site and the selectionmarker sequence to ensure in frame translation for the fusion protein.In some embodiments, the first SOI encodes an antibody. In someembodiments, the antibody is a single chain antibody, an antibody lightchain, an antibody heavy chain, a single-chain Fv fragment (scFv), or anFc fusion protein. In some embodiments, the second SOI encodes anantibody. In some embodiments, the antibody is a single chain antibody,an antibody light chain, an antibody heavy chain, a single-chain Fvfragment (scFv), or an Fc fusion protein. In certain embodiments theantibodies encoded by the first and second SOIs pair to form amultispecific, e.g., bispecific antibody.

Such two vector RMCE strategies allow for the introduction of eight ormore SOIs by incorporating the appropriate number of SOIs between eachpair of RRSs.

Both single-vector and two-vector RMCE allow for unidirectionalintegration of one or more donor DNA molecule(s) into a pre-determinedsite of a host cell genome, and precise exchange of a DNA cassettepresent on the donor DNA with a DNA cassette on the host genome wherethe integration site resides. The DNA cassettes are characterized by twoheterospecific RRSs flanking at least one selection marker (although incertain two-vector RMCE examples a “split selection marker” can be usedas outlined herein) and/or at least one exogenous SOI. RMCE involvesdouble recombination cross-over events, catalyzed by a recombinase,between the two heterospecific RRSs within the target genomic locus andthe donor DNA molecule. RMCE is designed to introduce a copy of the SOIor selection marker into the pre-determined locus of a host cell genome.Unlike recombination which involves just one cross-over event, RMCE canbe implemented such that prokaryotic vector sequences are not introducedinto the host cell genome, thus reducing and/or preventing unwantedtriggering of host immune or defense mechanisms. The RMCE procedure canbe repeated with multiple DNA cassettes.

In certain embodiments, targeted integration is achieved by onecross-over recombination event, wherein one exogenous nucleotidesequence comprising one RRS adjacent to at least one exogenous SOI or atleast one selection marker is integrated into a pre-determined site of ahost cell genome. In certain embodiments, targeted integration isachieved by one RMCE, wherein a DNA cassette comprising at least anexogenous SOI or at least one selection marker flanked by twoheterospecific RRSs is integrated into a pre-determined site of a hostcell genome. In certain embodiments, targeted integration is achieved bytwo RMCEs, wherein two different DNA cassettes, each comprising at leastan exogenous SOI or at least one selection marker flanked by twoheterospecific RRSs, are both integrated into a pre-determined site of ahost cell genome. In certain embodiments, targeted integration isachieved by multiple RMCEs, wherein DNA cassettes from multiple vectors,each comprising at least an exogenous SOI or at least one selectionmarker flanked by two heterospecific RRSs, are all integrated into apre-determined site of a host cell genome. In certain embodiments theselection marker can be partially encoded on the first the vector andpartially encoded on the second vector such that the integration of bothRMCEs allows for the expression of the selection marker. An example ofsuch a system is presented in FIG. 4.

In certain embodiments, targeted integration via recombinase-mediatedrecombination leads to a selection marker or one or more exogenous SOIintegrated into one or more pre-determined integration sites of a hostcell genome along with sequences from a prokaryotic vector. In certainembodiments, targeted integration via recombinase-mediated recombinationleads to selection marker or one or more exogenous SOI integrated intoone or more pre-determined integration sites of a host cell genome freeof sequences from a prokaryotic vector.

5.2 Targeted Integration Via Homologous Recombination, HDR, or NHEJ

The presently disclosed subject matter also relates to targetedintegration mediated by homologous recombination or by an exogenoussite-specific nuclease followed by HDR or NHEJ.

Homologous recombination is a recombination between DNA molecules thatshare extensive sequence homology. It can be used to direct error-freerepair of double-stranded DNA breaks and generates sequence variation ingametes during meiosis. Since homologous recombination involves theexchange of genetic information between two homologous DNA molecules, itdoes not alter the overall arrangement of the genes on a chromosome.During homologous recombination, a nick or break forms indouble-stranded DNA (dsDNA), followed by the invasion of a homologousdsDNA molecule by a single-stranded DNA end, pairing of homologoussequences, branch migration to form a Holliday junction, and finalresolution of the Holliday junction.

Double-strand break (DSB) is the most severe form of DNA damage andrepair of such DNA damage is essential for the maintenance of genomeintegrity in all organisms. There are two major repair pathways torepair DSBs. The first repair pathway is homology-directed repair (HDR)pathway and homologous recombination is the most common form of HDR.Since HDR requires the presence of homologous DNA present in the cell,this repair pathway is normally active in S and G2 phase of the cellcycle wherein newly replicated sister chromatids are available ashomologous templates. HDR is also a major repair pathway to repaircollapsed replication forks during DNA replication. HDR is considered asa relatively error-free repair pathway. The second repair pathway forDSBs is non-homologous end joining (NHEJ). NHEJ is a repair pathwaywherein the ends of a broken DNA are ligated together without therequirement for a homologous DNA template.

Targeted integration can be facilitated by exogenous site-specificnucleases followed by HDR. This is due to that the frequency ofhomologous recombination can be increased by introducing a DSB at aspecific target genomic site. In certain embodiments, an exogenousnuclease can be selected from the group consisting of a zinc fingernuclease (ZFN), a ZFN dimer, a transcription activator-like effectornuclease (TALEN), a TAL effector domain fusion protein, an RNA-guidedDNA endonuclease, an engineered meganuclease, and a clustered regularlyinterspaced short palindromic repeats (CRISPR)-associated (Cas)endonuclease.

CRISPR/Cas and TALEN systems are two genome editing tools that offer thebest ease of construction and high efficiency. CRISPR/Cas was identifiedas an immune defense mechanism of bacteria against invadingbacteriophages. Cas is a nuclease that, when guided by a synthetic guideRNA (gRNA), is capable of associating with a specific nucleotidesequence in a cell and editing the DNA in or around that nucleotidesequence, for instance by making one or more of a single-strand break, aDSB, and/or a point mutation. TALEN is an engineered site-specificnuclease, which is composed of the DNA-binding domain of TALE(transcription activator-like effectors) and the catalytic domain ofrestriction endonuclease FokI. By changing the amino acids present inthe highly variable residue region of the monomers of the DNA bindingdomain, different artificial TALENs can be created to target variousnucleotides sequences. The DNA binding domain subsequently directs thenuclease to the target sequences and creates a DSB.

Targeted integration via homologous recombination or HDR involves thepresence of homologous sequences to the integration site. In certainembodiments, the homologous sequences are present on a vector. Incertain embodiments, the homologous sequences are present on apolynucleotide.

In certain embodiments, a vector for targeted integration of exogenousnucleotide sequences into a host cell comprises nucleotide sequenceshomologous to an endogenous gene selected from the group consisting ofLOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, andXP_003512331.2, or to a sequence selected from SEQ ID Nos. 1-7 flankingat least one selection marker. In certain embodiments, a vector fortargeted integration of exogenous nucleotide sequences into a host cellcomprises nucleotide sequences homologous to an endogenous gene selectedfrom the group consisting of LOC107977062, LOC100768845, ITPR2,ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or to a sequence selectedfrom SEQ ID Nos. 1-7 flanking at least one selection marker and at leastone exogenous SOI. In certain embodiments, a vector for targetedintegration of exogenous nucleotide sequences into a host cell comprisesnucleotide sequences at least 50% homologous to a sequence selected fromSEQ ID Nos. 1-7 flanking a DNA cassette, wherein the DNA cassettecomprises at least one selection marker and at least one exogenous SOIflanked by two RRSs. In certain embodiments, a vector for targetedintegration of exogenous nucleotide sequences into a host cell comprisesnucleotide sequences at least 50% homologous to an endogenous geneselected from the group consisting of LOC107977062, LOC100768845, ITPR2,ERE67000.1, UBAP2, MTMR2, and XP_003512331.2 flanking a DNA cassette,wherein the DNA cassette comprises at least one selection marker and atleast one exogenous SOI flanked by two RRSs. In certain embodiments, thevector nucleotide sequences are at least about 50%, at least about 60%,at least about 70%, at least about 80%, at least about 90%, at leastabout 95%, at least about 99%, or at least about 99.9% homologous to anendogenous gene selected from the group consisting of LOC107977062,LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or toa sequence selected from SEQ ID Nos. 1-7. In certain embodiments, thevector is selected from the group consisting of an adenovirus vector, anadeno-associated virus vector, a lentivirus vector, a retrovirus vector,an integrating phage vector, a non-viral vector, a transposon and/ortransposase vector, an integrase substrate, and a plasmid.

In certain embodiments, a polynucleotide for targeted integration ofexogenous nucleotide sequences into a host cell comprises nucleotidesequences homologous to an endogenous gene selected from the groupconsisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2,MTMR2, and XP_003512331.2, or to a sequence selected from SEQ ID Nos.1-7 flanking at least one selection marker. In certain embodiments, apolynucleotide for targeted integration of exogenous nucleotidesequences into a host cell comprises nucleotide sequences homologous toan endogenous gene selected from the group consisting of LOC107977062,LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or toa sequence selected from SEQ ID Nos. 1-7 flanking at least one selectionmarker and at least one exogenous SOI. In certain embodiments, apolynucleotide for targeted integration of exogenous nucleotidesequences into a host cell comprises nucleotide sequences at least 50%homologous to a sequence selected from SEQ ID Nos. 1-7 flanking a DNAcassette, wherein the DNA cassette comprises at least one selectionmarker and at least one exogenous SOI flanked by two RRSs. In certainembodiments, a polynucleotide for targeted integration of exogenousnucleotide sequences into a host cell comprises nucleotide sequences atleast 50% homologous to an endogenous gene selected from the groupconsisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2,MTMR2, and XP_003512331.2 flanking a DNA cassette, wherein the DNAcassette comprises at least one selection marker and at least oneexogenous SOI flanked by two RRSs. In certain embodiments, the flankingnucleotide sequences are at least about 50%, at least about 60%, atleast about 70%, at least about 80%, at least about 90%, at least about95%, at least about 99%, or at least about 99.9% homologous to anendogenous gene selected from the group consisting of LOC107977062,LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, and XP_003512331.2, or toa sequence selected from SEQ ID Nos. 1-7.

In certain embodiments, homologous recombination is carried out withoutany accessory factors. In certain embodiments, homologous recombinationis facilitated by the presence of vectors that are capable ofintegration. In certain embodiments, an integrating vector is selectedfrom the group consisting of an adeno-associated virus vector, alentivirus vector, a retrovirus vector, and an integrating phage vector.

5.3 Regulated Targeted integration

There are many cases where protein expression levels are not optimalmainly because the encoded proteins are difficult-to-express. The lowexpression level of difficult-to-express proteins can have diverse anddifficult to identify causes. One possibility is the toxicity of theexpressed proteins in the host cells. In such cases, a regulatedexpression system can be used to express toxic proteins where thesequences of interest encoding the proteins are under the control of aninducible promoter. In these systems, expression of thedifficult-to-express proteins is only prompted when a regulator, e.g.,small molecule, such as, but not limited to, tetracycline or itsanalogue, doxycycline (DOX), is added to the culture. Regulating theexpression of toxic proteins could alleviate the toxic effects, allowingthe cultures to achieve the desired cell growth prior to production. Incertain embodiments, a regulated target integration (RTI) systemcomprises a SOI that is integrated into a specific locus, e.g., anexogenous nucleic acid sequence comprising one or more RRSs, and istranscribed under a regulated promoter operably linked thereto. Incertain embodiments, an RTI system can be used to determine theunderlying causes of low protein expression for a difficult-to-expressmolecule, such as, but not limited to, an antibody. In certainembodiments, the ability to selectively turn off the expression of a SOIin an RTI system can be used to link expression of a SOI to an observedadverse effect.

In certain embodiments, to minimize transcriptional and cell linevariability effects during the root cause analysis ofdifficult-to-express molecules, a regulated target integration (RTI)system can be used. For example, but not by way of limitation, theexpression of the SOI in a TI host can be triggered by addition to theculture of a regulator, e.g., doxycycline. In certain embodiments, theRTI vector utilizes a tetracycline-regulated promoter to express theSOI, which can be integrated into, e.g., an exogenous nucleic acidsequence comprising an RRS, which is itself integrated into anintegration site in the host cell's genome, allowing for regulatedexpression of the SOI.

In certain embodiments, the RTI system described in the presentdisclosure can be used to successfully determine the underlying cause(s)of low protein expression of an SOI, e.g., a therapeutic antibody, ascompared to control cell line. In certain embodiments, once the lowerrelative expression of a SOI, e.g., a therapeutic antibody, in a RTIcell line is confirmed, the intracellular accumulation and secretionlevels of the SOI can be evaluated by leveraging protein translationinhibitor treatments, e.g., Dox and cycloheximide.

5.4 Regulated Systems

The presently disclosed subject matter also relates to regulated systemsfor use in TI. For example, but not by way limitation, such regulationcan be based on gene switches for blocking or activating mRNA synthesisby regulated coupling of transcriptional repressors or activators toconstitutive or minimal promoters. In certain non-limiting embodiments,repression can be achieved by binding the repressor proteins, e.g.,where the proteins sterically block transcriptional initiation, or byactively repressing transcription through transcriptional silencers. Incertain non-limiting embodiments, activation of mammalian or viralenhancerless minimal promoters can be achieved by the regulated couplingto an activation domain.

In certain embodiments, the conditional coupling of transcriptionalrepressors or activators can be achieved by using allosteric proteinsthat bind the promoters in response to external stimuli. In certainembodiments, the conditional coupling of transcriptional repressors oractivators can be achieved by using intracellular receptors that arereleased from sequestrating proteins and, thus, can bind targetpromoters. In certain embodiments, the conditional coupling oftranscriptional repressors or activators can be achieved by usingchemically induced dimerizers.

In certain embodiments, the allosteric proteins used in the TI systemsof the present disclosure can be proteins that modulate transcriptionalactivity in response to antibiotics, bacterial quorum-sensingmessengers, catabolites, or to the cultivation parameters, such astemperature, e.g. cold or heat. In certain embodiments, such RTI systemscan be catabolite-based, e.g., where a bacterial repressor that controlscatabolic genes for alternative carbon sources has been transferred tomammalian cells. In certain embodiments, the repression of the targetpromoter can be achieved by cumate-responsive binding of the repressorCymR. In certain embodiments, the catabolite-based system can rely onthe activation of chimeric promoters by 6-hydroxynicotine-responsivebinding of the prokaryotic repressor HdnoR, fused to the Herpes simplexVP16 transactivation domain.

In certain embodiments the TI system can be a quorum-sensing-basedexpression system originated from prokaryotes that manage intra- andinter-population communication by quorum-sensing molecules. Thesequorum-sensing molecules bind to receptors in target cells, modulate thereceptors' affinity to cognate promoters leading to the initiation ofspecific regulon switches. In certain embodiments, the quorum-sensingmolecule can be the N-(3-oxo-octanoyl)-homoserine lactone in thepresence of which, the TraR-p65 fusion protein activates expression froma minimal promoter fused to the TraR-specific operator sequence. Incertain embodiments, the quorum-sensing molecule can be thebutyrolactone SCB1 (racemic2-(1′-hydroxy-6-methylheptyl)-3-(hydroxymethyl)-butanolide) in a systembased on the Streptomyces coelicolor A3(2) ScbR repressor that binds itscognate operator OScbR in the absence of the SCB1. In certainembodiments, the quorum-sensing molecule can be homoserine-derivedinducers used in a RTI system wherein Pseudomonas aeruginosaquorum-sensing repressors RhlR and LasR are fused to the SV40 T-antigennuclear localization sequence and the Herpes simplex VP16 domain and canactivate promoters containing specific operator sequences (las boxes).

In certain embodiments, the inducing molecules that modulate theallosteric proteins used in the RTI systems of the present disclosurecan be, but are not limited to, cumate, isopropyl-β-D-galactopyranoside(IPTG), macrolides, 6-hydroxynicotine, doxycycline, streptogramins,NADH, tetracycline.

In certain embodiments, the intracellular receptors used in the RTIsystems of the present disclosure can be cytoplasmic or nuclearreceptors. In certain embodiments, the RTI systems of the presentdisclosure can utilize the release of transcription factors fromsequestering and inhibiting proteins by using small molecules. Incertain embodiments, the RTI systems of the present disclosure can relyon steroid-regulation, wherein a hormone receptor is fused to a naturalor an artificial transcription factor that can be released from HSP90 inthe cytosol, migrate into the nucleus and activate selected promoters.In certain embodiments, mutant receptors can be used that are regulatedby synthetic steroid analogs in order to avoid crosstalk by endogenoussteroid hormones. In certain embodiments the receptors can be anestrogen receptor variant responsive to 4-hydroxytamoxifen or aprogesterone-receptor mutant inducible by RU486. In certain embodiments,the nuclear receptor-derived rosiglitazone-responsive transcriptionswitch based on the human nuclear peroxisome proliferator-activatedreceptor γ(PPARγ) can be used in the RTI systems of the presentdisclosure. In certain embodiments, a variant of steroid-responsivereceptors can be the RheoSwitch, that is based on a modifiedChoristoneura fumiferana ecdysone receptor and the mouse retinoid Xreceptor (RXR) fused to the Gal4 DNA binding domain and the VP16trans-activator. In the presence of synthetic ecdysone, the RheoSwitchvariant can bind and activate a minimal promoter fused to severalrepeats of the Gal4-response element.

In certain embodiments, the RTI systems disclosed herein can utilizechemically induced dimerization of a DNA-binding protein and atranscriptional activator for the activation of a minimal core promoterfused with a cognate operator. In certain embodiments, the RTI systemsdisclosed herein can utilize the rapamycin-regulated dimerization ofFKBP with FRB. In this system the FRB is fused to the p65trans-activator and FKBP is fused to a zinc finger domain specific forcognate operator sites placed upstream of an engineered minimalinterleukin-12 promoter. In certain embodiments, the FKBP can bemutated. In certain embodiments, the RTI systems disclosed herein canutilize bacterial gyrase B subunit (GyrB), where GyrB dimerizes in thepresence of the antibiotic coumermycin and dissociates with novobiocin.

In certain embodiments, the RTI systems of the present disclosure can beused for regulated siRNA expression. In certain embodiments, theregulated siRNA expression system can be a tetracycline, a macrolide, oran OFF- and ON-type QuoRex system. In certain embodiments, the RTIsystem can utilize a Xenopus terminal oligopyrimidine element (TOP),which blocks translational initiation by forming hairpin structures inthe 5′ untranslated region.

In certain embodiments, the RTI systems described in the presentdisclosure can utilize gas-phase controlled expression, e.g.,acetaldehyde-induced regulation (AIR) system. The AIR system can employthe Aspergillus nidulans AlcR transcription factor, which specificallyactivates the PAIR promoter assembled from AlcR-specific operators fusedto the minimal human cytomegalovirus promoter in the presence of gaseousor liquid acetaldehyde at nontoxic concentrations.

In certain embodiments, the RTI systems of the present disclosure canutilize a Tet-On or a Tet-Off system. In such systems, expression of aone or more SOIs can be regulated by tetracycline or its analogue,doxycycline.

In certain embodiments, the RTI system of the present disclosure canutilize a PIP-on or a PIP-off system. In such systems, the expression ofSOIs can be regulated by, e.g., pristinamycin, tetracycline and/orerythromycin.

6. Preparation and Use of TI Host Cells

The presently disclosed subject matter relates to methods for thetargeted integration of exogenous nucleotide sequences into a host cell.In certain embodiments, the methods relate to the integration of anexogenous nucleotide sequence into a host cell to produce a host cellsuitable for subsequent targeted integration of a SOI. In certainembodiments, said methods comprise recombinase-mediated recombination.In certain embodiments, said methods involve homologous recombination,HDR, and/or NHEJ.

6.1 Preparation of TI Host Cells Using Recombinase-MediatedRecombination

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a polypeptide of interest comprising:a) providing a TI host cell comprising an exogenous nucleotide sequenceintegrated at a site within a locus of the genome of the host cell,wherein the locus is at least about 90% homologous to SEQ ID Nos. 1-7,wherein the exogenous nucleotide sequence comprises two RRSs, flankingat least one first selection marker; b) introducing into the cellprovided in a) a vector comprising two RRSs matching the two RRSs on theintegrated exogenous nucleotide sequence and flanking at least oneexogenous SOI and at least one second selection marker; c) introducing arecombinase, wherein the recombinase recognizes the RRSs; and d)selecting for TI cells expressing the second selection marker to therebyisolate a TI host cell expressing the polypeptide of interest.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a polypeptide of interest comprising:a) providing a TI host cell comprising an exogenous nucleotide sequenceintegrated at a site within an endogenous gene selected from the groupconsisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2,MTMR2, XP_003512331.2, and at least about 90% homologous sequencesthereof, wherein the exogenous nucleotide sequence comprises two RRSs,flanking at least one first selection marker; b) introducing into thecell provided in a) a vector comprising two RRSs matching the two RRSson the integrated exogenous nucleotide sequence and flanking at leastone exogenous SOI and at least one second selection marker; c)introducing a recombinase, wherein the recombinase recognizes the RRSs;and d) selecting for TI cells expressing the second selection marker tothereby isolate a TI host cell expressing the polypeptide of interest.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a polypeptide of interest comprising:a) providing a TI host cell comprising an exogenous nucleotide sequenceintegrated at a site within a locus of the genome of the TI host cell,wherein the locus is at least about 90% homologous to SEQ ID Nos. 1-7,wherein the exogenous nucleotide sequence comprises a first DNA cassettecomprising two heterospecific RRSs, flanking at least one firstselection marker; b) introducing into the cell provided in a) a vectorcomprising a second DNA cassette comprising two heterospecific RRSsmatching the two RRSs on the integrated exogenous nucleotide sequenceand flanking at least one exogenous SOI and at least one secondselection marker; c) introducing a recombinase, wherein the recombinaserecognizes the RRSs and performs one RMCE; and d) selecting for TI cellsexpressing the second selection marker to thereby isolate a TI host cellexpressing the polypeptide of interest.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a polypeptide of interest comprising:a) providing a TI host cell comprising an exogenous nucleotide sequenceintegrated at a site within an endogenous gene selected from the groupconsisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2,MTMR2, XP_003512331.2, and at least about 90% homologous sequencesthereof, wherein the exogenous nucleotide sequence comprises a first DNAcassette comprising two heterospecific RRSs, flanking at least one firstselection marker; b) introducing into the cell provided in a) a vectorcomprising a second DNA cassette comprising two heterospecific RRSsmatching the two RRSs on the integrated exogenous nucleotide sequenceand flanking at least one exogenous SOI and at least one secondselection marker; c) introducing a recombinase, wherein the recombinaserecognizes the RRSs and performs one RMCE; and d) selecting for TI cellsexpressing the second selection marker to thereby isolate a TI host cellexpressing the polypeptide of interest.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a first and second polypeptide ofinterest (where the first and second polypeptides can be the same ordifferent) comprising: a) providing a TI host cell comprising anexogenous nucleotide sequence integrated at a site within a locus of thegenome of the host cell, wherein the locus is at least about 90%homologous to SEQ ID Nos. 1-7, wherein the exogenous nucleotide sequencecomprises a first and a second RRS flanking at least one first selectionmarker, and a third RRS located between the first and the second RRS,and all the RRSs are heterospecific; b) introducing into the cellprovided in a) a first vector comprising two RRSs matching the first andthe third RRS on the integrated exogenous nucleotide sequence andflanking at least one first exogenous SOI and at least one secondselection marker; c) introducing into the cell provided in a) a secondvector comprising two RRSs matching the second and the third RRS on theintegrated exogenous nucleotide sequence and flanking at least onesecond exogenous SOI; d) introducing one or more recombinases, whereinthe one or more recombinases recognize the RRSs; and e) selecting for TIcells expressing the second selection marker to thereby isolate a TIhost cell expressing the first and second polypeptides of interest. Incertain embodiments, rather than have the entire selection maker on thefirst vector, the first vector comprises a promoter sequence operablylinked to the codon ATG positioned flanked upstream by the first SOI anddownstream by an RRS; and the second vector comprises a selection markerlacking an ATG transcription start codon flanked upstream by an RRS anddownstream by the second SOI.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a first and second polypeptide ofinterest (where the first and second polypeptides can be the same ordifferent) comprising: a) providing a TI host cell comprising anexogenous nucleotide sequence integrated at a site within an endogenousgene selected from the group consisting of LOC107977062, LOC100768845,ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and at least about 90%homologous sequences thereof, wherein the exogenous nucleotide sequencecomprises a first and a second RRS flanking at least one first selectionmarker, and a third RRS located between the first and the second RRS,and all the RRSs are heterospecific; b) introducing into the cellprovided in a) a first vector comprising two RRSs matching the first andthe third RRS on the integrated exogenous nucleotide sequence andflanking at least one first exogenous SOI and at least one secondselection marker; c) introducing into the cell provided in a) a secondvector comprising two RRSs matching the second and the third RRS on theintegrated exogenous nucleotide sequence and flanking at least onesecond exogenous SOI; d) introducing one or more recombinases, whereinthe one or more recombinases recognize the RRSs; and e) selecting for TIcells expressing the second selection marker to thereby isolate a TIhost cell expressing the first and second polypeptides of interest. Incertain embodiments, rather than have the entire selection maker on thefirst vector, the first vector comprises a promoter sequence operablylinked to the codon ATG positioned flanked upstream by the first SOI anddownstream by an RRS; and the second vector comprises a selection markerlacking an ATG transcription start codon flanked upstream by an RRS anddownstream by the second SOI.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a first and second polypeptide ofinterest (where the first and second polypeptides can be the same ordifferent) comprising: a) providing a TI host cell comprising anexogenous nucleotide sequence integrated at a site within a locus of thegenome of the host cell, wherein the locus is at least about 90%homologous to SEQ ID Nos. 1-7, wherein the exogenous nucleotide sequencecomprises a first DNA cassette comprising a first and a second RRSflanking at least one first selection marker, and a third RRS locatedbetween the first and the second RRS, and all three RRSs areheterospecific; b) introducing into the cell provided in a) a firstvector comprising a second DNA cassette, wherein the second DNA cassettecomprises two heterospecific RRSs matching the first and the third RRSof the first DNA cassette and flanking at least one first exogenous SOIand at least one second selection marker; c) introducing into the cellprovided in a) a second vector comprising a third DNA cassette, whereinthe third DNA cassette comprises two heterospecific RRSs matching thesecond and the third RRS of the first DNA cassette and flanking at leastone second exogenous SOI; d) introducing one or more recombinases,wherein the one or more recombinases recognize the RRSs and perform twoRMCEs; and e) selecting for TI cells expressing the second selectionmarker to thereby isolate a TI host cell expressing the first and secondpolypeptides of interest. In certain embodiments, rather than have theentire selection maker on the first vector, the first vector comprises apromoter sequence operably linked to the codon ATG positioned flankedupstream by the first SOI and downstream by an RRS; and the secondvector comprises a selection marker lacking an ATG transcription startcodon flanked upstream by an RRS and downstream by the second SOI.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a first and second polypeptide ofinterest (where the first and second polypeptides can be the same ordifferent) comprising: a) providing a TI host cell comprising anexogenous nucleotide sequence integrated at a site within an endogenousgene selected from the group consisting of LOC107977062, LOC100768845,ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at leastabout 90% homologous thereto, wherein the exogenous nucleotide sequencecomprises a first DNA cassette comprising a first and a second RRSflanking at least one first selection marker, and a third RRS locatedbetween the first and the second RRS, and all three RRSs areheterospecific; b) introducing into the cell provided in a) a firstvector comprising a second DNA cassette, wherein the second DNA cassettecomprises two heterospecific RRSs matching the first and the third RRSof the first DNA cassette and flanking at least one first exogenous SOIand at least one second selection marker; c) introducing into the cellprovided in a) a second vector comprising a third DNA cassette, whereinthe third DNA cassette comprises two heterospecific RRSs matching thesecond and the third RRS of the first DNA cassette and flanking at leastone second exogenous SOI; d) introducing one or more recombinases,wherein the one or more recombinases recognize the RRSs and perform twoRMCEs; and e) selecting for TI cells expressing the second selectionmarker to thereby isolate a TI host cell expressing the first and secondpolypeptides of interest. In certain embodiments, rather than have theentire selection maker on the first vector, the first vector comprises apromoter sequence operably linked to the codon ATG positioned flankedupstream by the first SOI and downstream by an RRS; and the secondvector comprises a selection marker lacking an ATG transcription startcodon flanked upstream by an RRS and downstream by the second SOI.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a polypeptide of interest comprising:a) providing a TI host cell comprising an exogenous nucleotide sequenceintegrated at a site within a locus of the genome of the host cell,wherein the locus is at least about 90% homologous to a sequenceselected from SEQ ID Nos. 1-7, wherein the exogenous nucleotide sequencecomprises one RRS adjacent to at least one first selection marker; b)introducing into the cell provided in a) a vector comprising one RRSmatching the RRS on the integrated exogenous nucleotide sequence andadjacent to at least one exogenous SOI and at least one second selectionmarker; c) introducing a recombinase, wherein the recombinase recognizesthe RRSs; and d) selecting for TI cells expressing the second selectionmarker to thereby isolate a TI host cell expressing the polypeptide ofinterest.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a polypeptide of interest comprising:a) providing a TI host cell comprising an exogenous nucleotide sequenceintegrated at a site within an endogenous gene selected from the groupconsisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2,MTMR2, XP_003512331.2, and sequences at least about 90% homologousthereto, wherein the exogenous nucleotide sequence comprises one RRSadjacent to at least one first selection marker; b) introducing into thecell provided in a) a vector comprising one RRS matching the RRS on theintegrated exogenous nucleotide sequence and adjacent to at least oneexogenous SOI and at least one second selection marker; c) introducing arecombinase, wherein the recombinase recognizes the RRSs; and d)selecting for TI cells expressing the second selection marker to therebyisolate a TI host cell expressing the polypeptide of interest.

The presently disclosed subject matter also relates to methods ofproducing a polypeptide of interest comprising: a) providing a TI hostcell described herein; b) culturing the TI host cell in a) underconditions suitable for expressing the SOI and recovering a polypeptideof interest therefrom.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells suitable for subsequent targeted integrationcomprising: a) providing a TI host cell comprising an exogenousnucleotide sequence integrated at a site within a locus of the genome ofthe host cell, wherein the locus is at least about 90% homologous to asequence selected from SEQ ID Nos. 1-7, wherein the exogenous nucleotidesequence comprises two RRSs flanking at least one exogenous SOI and atleast one first selection marker; b) introducing into the cell providedin a) a vector comprising two RRSs matching the two RRSs on theintegrated exogenous nucleotide sequence and flanking at least onesecond selection marker; c) introducing a recombinase, wherein therecombinase recognizes the RRSs; and d) selecting for TI cellsexpressing the second selection marker to thereby isolate a TI host cellsuitable for subsequent targeted integration.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells suitable for subsequent targeted integrationcomprising: a) providing a TI host cell comprising an exogenousnucleotide sequence integrated at a site within an endogenous geneselected from the group consisting of LOC107977062, LOC100768845, ITPR2,ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about90% homologous thereto, wherein the exogenous nucleotide sequencecomprises two RRSs flanking at least one exogenous SOI and at least onefirst selection marker; b) introducing into the cell provided in a) avector comprising two RRSs matching the two RRSs on the integratedexogenous nucleotide sequence and flanking at least one second selectionmarker; c) introducing a recombinase, wherein the recombinase recognizesthe RRSs; and d) selecting for TI cells expressing the second selectionmarker to thereby isolate a TI host cell suitable for subsequenttargeted integration.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells suitable for subsequent targeted integrationcomprising: a) providing a TI host cell comprising an exogenousnucleotide sequence integrated at a site within a locus of the genome ofthe host cell, wherein the locus is at least about 90% homologous to asequence selected from SEQ ID Nos. 1-7, wherein the exogenous nucleotidesequence comprises a first and a second RRS flanking at least oneexogenous SOI and at least one first selection marker; b) introducinginto the cell provided in a) a vector comprising three RRSs, wherein thefirst RRS of the vector matches the first RRS on the integratedexogenous nucleotide sequence, the second RRS of the vector matches thesecond RRS on the integrated exogenous nucleotide sequence, and at leastone second selection marker located between the first and the secondRRS; c) introducing a recombinase, wherein the recombinase recognizesthe first and the second RRS on both the vector and the integratedexogenous nucleotide sequence; and d) selecting for TI host cellsexpressing the second selection marker to thereby isolate a TI host cellsuitable for subsequence targeted integration.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells suitable for subsequent targeted integrationcomprising: a) providing a TI host cell comprising an exogenousnucleotide sequence integrated at a site within an endogenous geneselected from the group consisting of LOC107977062, LOC100768845, ITPR2,ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about90% homologous thereto, wherein the exogenous nucleotide sequencecomprises a first and a second RRS flanking at least one exogenous SOIand at least one first selection marker; b) introducing into the cellprovided in a) a vector comprising three RRSs, wherein the first RRS ofthe vector matches the first RRS on the integrated exogenous nucleotidesequence, the second RRS of the vector matches the second RRS on theintegrated exogenous nucleotide sequence, and at least one secondselection marker located between the first and the second RRS; c)introducing a recombinase, wherein the recombinase recognizes the firstand the second RRS on both the vector and the integrated exogenousnucleotide sequence; and d) selecting for TI host cells expressing thesecond selection marker to thereby isolate a TI host cell suitable forsubsequent targeted integration.

6.2 Methods for Targeted Modification of a Host Cell Using HomologousRecombination, HDR, or NHEJ

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a polypeptide of interest comprising:a) providing a TI host cell comprising a locus of the genome of the hostcell, wherein the locus is at least about 90% homologous to SEQ ID Nos.1-7; b) introducing a vector into the TI host cell, wherein the vectorcomprises nucleotide sequences at least 50% homologous to a sequenceselected from SEQ ID No. 1-7 flanking a DNA cassette, wherein the DNAcassette comprises at least one selection marker and at least oneexogenous SOI; c) selecting for the selection marker to isolate a TIhost cell with the SOI integrated in the locus of the genome, andexpressing the polypeptide of interest. In certain embodiments, the DNAcassette of the vector further comprises at least one selection markerand at least one exogenous SOI flanked by two RRSs.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells to express a polypeptide of interest comprisingg: a) providing a TI host cell comprising a locus of the genome of thehost cell, wherein the locus is at least about 90% homologous to asequence selected from SEQ ID Nos. 1-7; b) introducing a polynucleotideinto the host cell, wherein the polynucleotide comprises nucleotidesequences at least 50% homologous to a sequence selected from SEQ ID No.1-7 flanking a DNA cassette, wherein the DNA cassette comprises at leastone selection marker and at least one exogenous SOI; c) selecting forthe selection marker to isolate a TI host cell with the SOI integratedin the locus of the genome, and expressing the polypeptide of interest.In certain embodiments, the DNA cassette of the vector further comprisesat least one selection marker and at least one exogenous SOI flanked bytwo RRSs.

In certain embodiments, the homologous recombination is facilitated byan integrating vector. In certain embodiments, a vector is selected fromthe group consisting of an adenovirus vector, an adeno-associated virusvector, a lentivirus vector, a retrovirus vector, an integrating phagevector, a non-viral vector, a transposon and/or transposase vector, anintegrase substrate, and a plasmid. In certain embodiments, thetransposon can be a PiggyBac (PB) transposon system.

In certain embodiments, the integration is promoted by an exogenousnuclease. In certain embodiments, the exogenous nuclease is selectedfrom the group consisting of a zinc finger nuclease (ZFN), a ZFN dimer,a transcription activator-like effector nuclease (TALEN), a TAL effectordomain fusion protein, an RNA-guided DNA endonuclease, an engineeredmeganuclease, and a clustered regularly interspaced short palindromicrepeats (CRISPR)-associated (Cas) endonuclease.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells suitable for subsequent targeted integrationcomprising: a) providing a TI host cell comprising a locus of the genomeof the host cell, wherein the locus is at least about 90% homologous toa sequence selected from SEQ ID Nos. 1-7; b) introducing a vector intothe TI host cell, wherein the vector comprises nucleotide sequences atleast 50% homologous to a sequences selected from SEQ ID Nos. 1-7flanking a DNA cassette, wherein the DNA cassette comprises at least oneselection marker flanked by two RRSs; c) selecting for the selectionmarker to isolate a TI host cell suitable for subsequent targetedintegration.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells suitable for subsequent targeted integrationcomprising: a) providing a TI host cell comprising a locus of the genomeof the host cell, wherein the locus is at least about 90% homologous toa sequence selected from SEQ ID Nos. 1-7; b) introducing apolynucleotide into the TI host cell, wherein the polynucleotidecomprises nucleotide sequences at least 50% homologous to a sequenceselected from SEQ ID Nos. 1-7 flanking a DNA cassette, wherein the DNAcassette comprises at least one selection marker flanked by two RRSs; c)selecting for the selection marker to isolate a TI host cell suitablefor subsequent targeted integration.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells suitable for subsequent targeted integrationcomprising: a) providing a TI host cell comprising a locus of the genomeof the host cell, wherein the locus is at least about 90% homologous toa sequence selected from SEQ ID Nos. 1-7; b) introducing a vector intothe host cell, wherein the vector comprises nucleotide sequences atleast 50% homologous to a sequence selected from SEQ ID Nos. 1-7flanking a DNA cassette, wherein the DNA cassette comprises three RRSs,wherein the third RRS and at least one selection marker is locatedbetween the first and the second RRS; and c) selecting for the selectionmarker to isolate a TI host cell suitable for subsequent targetedintegration.

In certain embodiments, the present disclosure provides methods forpreparing TI host cells suitable for subsequent targeted integrationcomprising: a) providing a TI host cell comprising a locus of the genomeof the host cell, wherein the locus is at least about 90% homologous toa sequence selected from SEQ ID Nos. 1-7; b) introducing apolynucleotide into the host cell, wherein the polynucleotide comprisesnucleotide sequences at least 50% homologous to a sequence selected fromSEQ ID Nos. 1-7 flanking a DNA cassette, wherein the DNA cassettecomprises three RRSs, wherein the third RRS and at least one selectionmarker is located between the first and the second RRS; and c) selectingfor the selection marker to isolate a TI host cell suitable forsubsequent targeted integration.

In certain embodiments, the present disclosure provides methods forpreparing a TI host cell expressing at least one polypeptide of interestcomprising: a) providing a TI host cell comprising at least oneexogenous nucleotide sequence integrated at a site within one or moreloci of the genome of the TI host cell, wherein the one or more loci areat least about 90% homologous to a sequence selected from SEQ ID Nos.1-7, wherein the at least one exogenous nucleotide sequence comprisestwo RRSs, flanking at least one first selection marker; b) introducinginto the cell provided in a) a vector comprising two RRSs matching thetwo RRSs on the integrated exogenous nucleotide sequence and flanking atleast one exogenous SOI and at least one second selection marker; c)introducing a recombinase or a nucleic acid encoding a recombinase,wherein the recombinase recognizes the RRSs; and selecting for TI cellsexpressing the second selection marker to thereby isolate a TI host cellexpressing the at least one polypeptide of interest.

In certain embodiments, the present disclosure provides methods forpreparing a TI host cell expressing at least one first and secondpolypeptide of interest (where the first and second polypeptides can bethe same or different) comprising: a) providing a TI host cellcomprising at least one exogenous nucleotide sequence integrated at asite within one or more loci of the genome of the host cell, wherein oneor more loci are at least about 90% homologous to a sequence selectedfrom SEQ ID Nos. 1-7, wherein the exogenous nucleotide sequencecomprises a first and a second RRS flanking at least one first selectionmarker, and a third RRS located between the first and the second RRS,and all the RRSs are heterospecific; b) introducing into the cellprovided in a) a first vector comprising two RRSs matching the first andthe third RRS on the at least one integrated exogenous nucleotidesequence and flanking at least one first exogenous SOI and at least onesecond selection marker; c) introducing into the cell provided in a) asecond vector comprising two RRSs matching the second and the third RRSon the at least one integrated exogenous nucleotide sequence andflanking at least one second exogenous SOI; d) introducing one or morerecombinases, or one or more nucleic acids encoding one or morerecombinases, wherein the one or more recombinases recognize the RRSs;and e) selecting for TI cells expressing the second selection marker tothereby isolate a TI host cell expressing the at least one first andsecond polypeptides of interest. In certain embodiments, rather thanhave the entire selection maker on the first vector, the first vectorcomprises a promoter sequence operably linked to the codon ATGpositioned flanked upstream by the first SOI and downstream by an RRS;and the second vector comprises a selection marker lacking an ATGtranscription start codon flanked upstream by an RRS and downstream bythe second SOI.

In certain embodiments, the present disclosure provides methods forpreparing a TI host cell expressing a polypeptide of interestcomprising: a) providing a TI host cell comprising at least oneexogenous nucleotide sequence integrated at a site within one or moreloci of the genome of the TI host cell, wherein the one or more loci areat least about 90% homologous to a sequence selected from SEQ ID Nos.1-7, wherein the exogenous nucleotide sequence comprises one or moreRRSs; b) introducing into the cell provided in a) a vector comprisingone or more RRSs matching the one or more RRSs on the integratedexogenous nucleotide sequence and flanking at least one exogenous SOIoperably linked to a regulatable promoter; c) introducing a recombinaseor a nucleic acid encoding a recombinase, wherein the recombinaserecognizes the RRSs; and d) selecting for TI cells expressing theexogenous SOI in the presence of an inducer to thereby isolate a TI hostcell expressing the polypeptide of interest.

In certain embodiments, the present disclosure provides methods forexpressing a polypeptide of interest comprising: a) providing a hostcell comprising at least one exogenous SOI flanked by two RRSs and aregulatable promoter integrated within a locus of the genome of the hostcell, wherein the locus is at least about 90% homologous to a sequenceselected from SEQ ID Nos. 1-7; and b) culturing the cell underconditions suitable for expressing the SOI and recovering a polypeptideof interest therefrom.

In certain embodiments, the present disclosure provides methods forpreparing a TI host cell expressing a first and second polypeptide ofinterest (where the first and second polypeptides can be the same ordifferent) comprising: a) providing a TI host cell comprising anexogenous nucleotide sequence integrated at a site within a locus of thegenome of the host cell, wherein the locus is at least about 90%homologous to a sequence selected from SEQ ID Nos. 1-7, wherein theexogenous nucleotide sequence comprises a first, second RRS and a thirdRRS located between the first and the second RRS, and all the RRSs areheterospecific; b) introducing into the cell provided in a) a firstvector comprising two RRSs matching the first and the third RRS on theintegrated exogenous nucleotide sequence and flanking at least one firstexogenous SOI operably linked to a regulatable promoter; c) introducinginto the cell provided in a) a second vector comprising two RRSsmatching the second and the third RRS on the integrated exogenousnucleotide sequence and flanking at least one second SOI operably linkedto a regulatable promoter; d) introducing one or more recombinases, orone or more nucleic acids encoding one or more recombinases, wherein theone or more recombinases recognize the RRSs; and e) selecting for TIcells expressing the at least first and second exogenous SOIs in thepresence of an inducer to thereby isolate a TI host cell expressing thepolypeptides of interest. In certain embodiments, rather than have theentire selection maker on the first vector, the first vector comprises apromoter sequence operably linked to the codon ATG positioned flankedupstream by the first SOI and downstream by an RRS; and the secondvector comprises a selection marker lacking an ATG transcription startcodon flanked upstream by an RRS and downstream by the second SOI.

7. Products

The host cells of the present disclosure can be used for the expressionof any molecule of interest, e.g., a polypeptide of interest. In certainembodiments, the host cells of the present disclosure can be used forthe expression of polypeptides, e.g., mammalian polypeptides.Non-limiting examples of such polypeptides include hormones, receptors,fusion proteins, regulatory factors, growth factors, complement systemfactors, enzymes, clotting factors, anti-clotting factors, kinases,cytokines, CD proteins, interleukins, therapeutic proteins, diagnosticproteins and antibodies. In some embodiments, the antibody is amonoclonal antibody. In some embodiments, the antibody is a therapeuticantibody. In some embodiments, the antibody is a diagnostic antibody. Insome embodiments, the antibody is a human antibody. In some embodiments,the antibody is a humanized antibody.

In certain embodiments, examples of polypeptides encompassed within thedefinition herein include mammalian polypeptides, such as, e.g., renin;a growth hormone, including human growth hormone and bovine growthhormone; growth hormone releasing factor; parathyroid hormone; thyroidstimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A-chain;insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin;luteinizing hormone; glucagon; leptin; clotting factors such as factorVIIIC, factor IX, tissue factor, and von Willebrands factor;anti-clotting factors such as Protein C; atrial natriuretic factor; lungsurfactant; a plasminogen activator, such as urokinase or human urine ortissue-type plasminogen activator (t-PA); bombesin; thrombin;hematopoietic growth factor; tumor necrosis factor-alpha and -beta; atumor necrosis factor receptor such as death receptor κ and CD120;TNF-related apoptosis-inducing ligand (TRAIL); B-cell maturation antigen(BCMA); B-lymphocyte stimulator (BLyS); a proliferation-inducing ligand(APRIL); enkephalinase; RANTES (regulated on activation normally T-cellexpressed and secreted); human macrophage inflammatory protein(MIP-1-alpha); a serum albumin such as human serum albumin;Muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;prorelaxin; mouse gonadotropin-associated peptide; a microbial protein,such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyte associatedantigen (CTLA), such as CTLA-4; inhibin; activin; platelet-derivedendothelial cell growth factor (PD-ECGF); a vascular endothelial growthfactor family protein (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, and P1GF);a platelet-derived growth factor (PDGF) family protein (e.g., PDGF-A,PDGF-B, PDGF-C, PDGF-D, and dimers thereof); fibroblast growth factor(FGF) family such as aFGF, bFGF, FGF4, and FGF9; epidermal growth factor(EGF); receptors for hormones or growth factors such as a VEGFreceptor(s) (e.g., VEGFR1, VEGFR2, and VEGFR3), epidermal growth factor(EGF) receptor(s) (e.g., ErbB1, ErbB2, ErbB3, and ErbB4 receptor),platelet-derived growth factor (PDGF) receptor(s) (e.g., PDGFR-α andPDGFR-β), and fibroblast growth factor receptor(s); TIE ligands(Angiopoietins, ANGPT1, ANGPT2); Angiopoietin receptor such as TIE1 andTIE2; protein A or D; rheumatoid factors; a neurotrophic factor such asbone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6(NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-b;transforming growth factor (TGF) such as TGF-alpha and TGF-beta,including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5; insulin-like growthfactor-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I),insulin-like growth factor binding proteins (IGFBPs); CD proteins suchas CD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors;immunotoxins; a bone morphogenetic protein (BMP); a chemokine such asCXCL12 and CXCR4; an interferon such as interferon-alpha, -beta, and-gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, andG-CSF; a cytokine such as interleukins (ILs), e.g., IL-1 to IL-10;midkine; superoxide dismutase; T-cell receptors; surface membraneproteins; decay accelerating factor; viral antigen such as, for example,a portion of the AIDS envelope; transport proteins; homing receptors;addressins; regulatory proteins; integrins such as CD11a, CD11b, CD11c,CD18, an ICAM, VLA-4 and VCAM; ephrins; Bv8; Delta-like ligand 4 (DLL4);Del-1; BMP9; BMP10; Follistatin; Hepatocyte growth factor (HGF)/scatterfactor (SF); Alkl; Robo4; ESM1; Perlecan; EGF-like domain, multiple 7(EGFL7); CTGF and members of its family; thrombospondins such asthrombospondin1 and thrombospondin2; collagens such as collagen IV andcollagen XVIII; neuropilins such as NRP1 and NRP2; Pleiotrophin (PTN);Progranulin; Proliferin; Notch proteins such as Notch1 and Notch4;semaphorins such as Sema3A, Sema3C, and Sema3F; a tumor associatedantigen such as CA125 (ovarian cancer antigen); immunoadhesins; andfragments and/or variants of any of the above-listed polypeptides aswell as antibodies, including antibody fragments, binding to one or moreprotein, including, for example, any of the above-listed proteins.

In certain embodiments, the polypeptide of interest is a bi-specific,tri-specific or multi-specific polypeptide, e.g. a bi-specific antibody.Various molecular formats for multispecific antibodies are known in theart and are included herein (see e.g., Spiess et al., Mol Immunol 67(2015) 95-106). A particular type of multispecific antibodies, alsoincluded herein, are bispecific antibodies designed to simultaneouslybind to a surface antigen on a target cell, e.g., a tumor cell, and toan activating, invariant component of the T cell receptor (TCR) complex,such as CD3, for retargeting of T cells to kill target cells. Otherexamples of bispecific antibody formats include, but are not limited to,the so-called “BiTE” (bispecific T cell engager) molecules wherein twoscFv molecules are fused by a flexible linker (see, e.g., WO2004/106381, WO 2005/061547, WO 2007/042261, and WO 2008/119567,Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)); diabodies(Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof,such as tandem diabodies (“TandAb”; Kipriyanov et al., J Mol Biol 293,41-56 (1999)); “DART” (dual affinity retargeting) molecules which arebased on the diabody format but feature a C-terminal disulfide bridgefor additional stabilization (Johnson et al., J Mol Biol 399, 436-449(2010)), and so-called triomabs, which are whole hybrid mouse/rat IgGmolecules (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467(2010)). Particular T cell bispecific antibody formats included hereinare described in WO 2013/026833, WO 2013/026839, WO 2016/020309; Bacacet al., Oncoimmunology 5(8) (2016) e1203498.

In certain embodiments, the host cells of the present disclosure can beused for the expression of chaperones, protein modifying enzymes, shRNA,gRNA or other proteins or peptides while expressing a therapeuticprotein or molecule of interest constitutively or regulated.

In some embodiments, the polypeptide expressed by the host cells of thepresent disclosure may bind to, or interact with, any protein,including, without limitation, cytokines, cytokine-related proteins, andcytokine receptors selected from the group consisting of 8MPI, 8MP2,8MP38 (GDFIO), 8MP4, 8MP6, 8MP8, CSFI (M-CSF), CSF2 (GM-CSF), CSF3(G-CSF), EPO, FGF1 (aFGF), FGF2 (PFGF), FGF3 (int-2), FGF4 (HST), FGF5,FGF6 (HST-2), FGF7 (KGF), FGF9, FGF1 0, FGF11, FGF12, FGF12B, FGF14,FGF16, FGF17, FGF19, FGF20, FGF21, FGF23, IGF1, IGF2, IFNA1, IFNA2,IFNA4, IFNA5, IFNA6, IFNA7, IFN81, IFNG, IFNWI, FEL1, FEL1 (EPSELON),FEL1 (ZETA), IL 1A, IL 1n, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL 11, IL 12A, IL 12B, IL 13, IL 14, IL 15, IL 16, IL 17, IL 17B, IL18, IL 19, IL20, IL22, IL23, IL24, IL25, IL26, IL27, IL28A, IL28B, IL29,IL30, PDGFA, PDGFB, TGFA, TGFB1, TGFB2, TGFBb3, LTA (TNF-0), LTB, TNF(TNF-α), TNFSF4 (OX40 ligand), TNFSF5 (CD40 ligand), TNFSF6 (FasL),TNFSF7 (CD27ligand), TNFSF8 (CD30ligand), TNFSF9 (4-1 BB ligand),TNFSF10 (TRAIL), TNFSF11 (TRANCE), TNFSF12 (APO3L), TNFSF13 (April),TNFSF13B, TNFSF14 (HVEM-L), TNFSF15 (VEGI), TNFSF18, HGF (VEGFD), VEGF,VEGFB, VEGFC, IL1R1, IL1R2, IL1RL1, IL1RL2, IL2RA, IL2RB, IL2RG, IL3RA,IL4R, IL5RA, IL6R, IL7R, IL8RA, IL8RB, IL9R, ILORA, IL10RB, IL 11RA,IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL15RA, IL17R, IL18R1, IL20RA,IL21R, IL22R, IL1HY1, IL1RAP, IL1RAPL1, IL1RAPL2, IL1RN, IL6ST, IL18BP,IL8RAP, IL22RA2, AIF1, HGF, LEP (leptin), PTN, and THPO.k.

In some embodiments, the polypeptide expressed by the host cells of thepresent disclosure may bind to, or interact with, a chemokine, chemokinereceptor, or a chemokine-related protein selected from the groupconsisting of CCLI (1-309), CCL2 (MCP-1/MCAF), CCL3 (MIP-Iα), CCL4(MIP-Iβ), CCL5 (RANTES), CCL7 (MCP-3), CCL8 (mcp-2), CCL11 (eotaxin),CCL 13 (MCP-4), CCL 15 (MIP-Iδ), CCL 16 (HCC-4), CCL 17 (TARC), CCL 18(PARC), CCL 19 (MDP-3b), CCL20 (MIP-3α), CCL21 (SLC/exodus-2), CCL22(MDC/STC-1), CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK),CCL26 (eotaxin-3), CCL27 (CTACK/ILC), CCL28, CXCLI (GROI), CXCL2 (GR02),CXCL3 (GR03), CXCL5 (ENA-78), CXCL6 (GCP-2), CXCL9 (MIG), CXCL 10 (IP10), CXCL 11 (1-TAC), CXCL 12 (SDFI), CXCL 13, CXCL 14, CXCL 16, PF4(CXCL4), PPBP (CXCL7), CX3CL 1 (SCYDI), SCYEI, XCLI (lymphotactin), XCL2(SCM-Iβ), BLRI (MDR15), CCBP2 (D6/JAB61), CCRI (CKRI/HM145), CCR2(mcp-IRB IRA), CCR3 (CKR3/CMKBR3), CCR4, CCR5 (CMKBR5/ChemR13), CCR6(CMKBR6/CKR-L3/STRL22/DRY6), CCR7 (CKR7/EBII), CCR8(CMKBR8/TER1/CKR-L1), CCR9 (GPR-9-6), CCRL1 (VSHK1), CCRL2 (L-CCR), XCR1(GPR5/CCXCR1), CMKLR1, CMKOR1 (RDC1), CX3CR1 (V28), CXCR4, GPR2 (CCR10),GPR31, GPR81 (FKSG80), CXCR3 (GPR9/CKR-L2), CXCR6 (TYMSTR/STRL33/Bonzo),HM74, IL8RA (IL8Ra), IL8RB (IL8Rβ), LTB4R (GPR16), TCP10, CKLFSF2,CKLFSF3, CKLFSF4, CKLFSF5, CKLFSF6, CKLFSF7, CKLFSF8, BDNF, C5, C5R1,CSF3, GRCC10 (C10), EPO, FY (DARC), GDF5, HDF1, HDF1α, DL8, PRL, RGS3,RGS13, SDF2, SLIT2, TLR2, TLR4, TREM1, TREM2, and VHL. In someembodiments, the polypeptide expressed by the host cells of the presentdisclosure may bind to, or interact with, 0772P (CA125, MUC16) (i.e.,ovarian cancer antigen), ABCF1; ACVR1; ACVR1B; ACVR2; ACVR2B; ACVRL1;ADORA2A; Aggrecan; AGR2; AICDA; AIF1; AIG1; AKAP1; AKAP2; AMH; AMHR2;amyloid beta; ANGPTL; ANGPT2; ANGPTL3; ANGPTL4; ANPEP; APC; APOC1; AR;ASLG659; ASPHD1 (aspartate beta-hydroxylase domain containing 1;LOC253982); AZGP1 (zinc-a-glycoprotein); B7.1; B7.2; BAD; BAFF-R (Bcell-activating factor receptor, BLyS receptor 3, BR3; BAG1; BAIl; BCL2;BCL6; BDNF; BLNK; BLRI (MDR15); BMP1; BMP2; BMP3B (GDF10); BMP4; BMP6;BMP8; BMPR1A; BMPR1B (bone morphogenic protein receptor-type IB); BMPR2;BPAG1 (plectin); BRCA1; Brevican; C19orf10 (IL27w); C3; C4A; C5; C5R1;CANT1; CASP1; CASP4; CAV1; CCBP2 (D6/JAB61); CCL1 (1-309); CCL11(eotaxin); CCL13 (MCP-4); CCL15 (MIP16); CCL16 (HCC-4); CCL17 (TARC);CCL18 (PARC); CCL19 (MIP-30); CCL2 (MCP-1); MCAF; CCL20 (MIP-3a); CCL21(MTP-2); SLC; exodus-2; CCL22 (MDC/STC-1); CCL23 (MPIF-1); CCL24(MPIF-2/eotaxin-2); CCL25 (TECK); CCL26 (eotaxin-3); CCL27 (CTACK/ILC);CCL28; CCL3 (MTP-Ia); CCL4 (MDP-I0); CCL5(RANTES); CCL7 (MCP-3); CCL8(mcp-2); CCNA1; CCNA2; CCND1; CCNE1; CCNE2; CCR1 (CKRI/HM145); CCR2(mcp-IRP/RA); CCR3 (CKR/CMKBR3); CCR4; CCR5 (CMKBR5/ChemR13); CCR6(CMKBR6/CKR-L3/STRL22/DRY6); CCR7 (CKBR7/EBI1); CCR8(CMKBR8/TER1/CKR-L1); CCR9 (GPR-9-6); CCRL1 (VSHK1); CCRL2 (L-CCR);CD164; CD19; CD1C; CD20; CD200; CD22 (B-cell receptor CD22-B isoform);CD24; CD28; CD3; CD37; CD38; CD3E; CD3G; CD3Z; CD4; CD40; CD40L; CD44;CD45RB; CD52; CD69; CD72; CD74; CD79A (CD79a, immunoglobulin-associatedalpha, a B cell-specific protein); CD79B; CDS; CD80; CD81; CD83; CD86;CDH1 (E-cadherin); CDH10; CDH12; CDH13; CDH18; CDH19; CDH20; CDH5; CDH7;CDH8; CDH9; CDK2; CDK3; CDK4; CDK5; CDK6; CDK7; CDK9; CDKN1A(p21/WAF1/Cip1); CDKN1B (p27/Kipl); CDKN1C; CDKN2A (P16NK4a); CDKN2B;CDKN2C; CDKN3; CEBPB; CER1; CHGA; CHGB; Chitinase; CHST10; CKLFSF2;CKLFSF3; CKLFSF4; CKLFSF5; CKLFSF6; CKLFSF7; CKLFSF8; CLDN3; CLDN7(claudin-7); CLL-1 (CLEC12A, MICL, and DCAL2); CLN3; CLU (clusterin);CMKLR1; CMKOR1 (RDC1); CNR1; COL 18A1; COL1A1; COL4A3; COL6A1;complement factor D; CR2; CRP; CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1,teratocarcinoma-derived growth factor); CSFI (M-CSF); CSF2 (GM-CSF);CSF3 (GCSF); CTLA4; CTNNB1 (b-catenin); CTSB (cathepsin B); CX3CL1(SCYDI); CX3CR1 (V28); CXCL1 (GRO1); CXCL10 (IP-10); CXCL11(I-TAC/IP-9); CXCL12 (SDF1); CXCL13; CXCL14; CXCL16; CXCL2 (GRO2); CXCL3(GRO3); CXCL5 (ENA-78/LIX); CXCL6 (GCP-2); CXCL9 (MIG); CXCR3(GPR9/CKR-L2); CXCR4; CXCR5 (Burkitt's lymphoma receptor 1, a Gprotein-coupled receptor); CXCR6 (TYMSTR/STRL33/Bonzo); CYBS; CYC1;CYSLTR1; DAB2IP; DES; DKFZp451J0118; DNCLI; DPP4; E16 (LAT1, SLC7A5);E2F1; ECGF1; EDG1; EFNA1; EFNA3; EFNB2; EGF; EGFR; ELAC2; ENG; ENOl;ENO2; ENO3; EPHB4; EphB2R; EPO; ERBB2 (Her-2); EREG; ERK8; ESR1; ESR2;ETBR (Endothelin type B receptor); F3 (TF); FADD; FasL; FASN; FCER1A;FCER2; FCGR3A; FcRH1 (Fc receptor-like protein 1); FcRH2 (IFGP4, IRTA4,SPAP1A (SH2 domain containing phosphatase anchor protein 1a), SPAPIB,SPAPIC); FGF; FGF1 (aFGF); FGF10; FGF11; FGF12; FGF12B; FGF13; FGF14;FGF16; FGF17; FGF18; FGF19; FGF2 (bFGF); FGF20; FGF21; FGF22; FGF23;FGF3 (int-2); FGF4 (HST); FGF5; FGF6 (HST-2); FGF7 (KGF); FGF8; FGF9;FGFR; FGFR3; FIGF (VEGFD); FEL (EPSILON); FIL1 (ZETA); FLJ12584;FLJ25530; FLRTI (fibronectin); FLT1; FOS; FOSL1 (FRA-1); FY (DARC);GABRP (GABAa); GAGEB1; GAGEC1; GALNAC4S-6ST; GATA3; GDF5; GDNF-Ral (GDNFfamily receptor alpha 1; GFRA1; GDNFR; GDNFRA; RETL1; TRNR1; RET1L;GDNFR-alphal; GFR-ALPHA-1); GEDA; GFI1; GGT1; GM-CSF; GNASI; GNRHI; GPR2(CCR10); GPR19 (G protein-coupled receptor 19; Mm.4787); GPR31; GPR44;GPR54 (KISS1 receptor; KISSIR; GPR54; HOT7T175; AXOR12); GPR81 (FKSG80);GPR172A (G protein-coupled receptor 172A; GPCR41; FLJ11856;D15Ertd747e); GRCCIO (C10); GRP; GSN (Gelsolin); GSTP1; HAVCR2; HDAC4;HDAC; HDAC7A; HDAC9; HGF; HIF1A; HOPi; histamine and histaminereceptors; HLA-A; HLA-DOB (Beta subunit of MHC class II molecule (Iaantigen); HLA-DRA; HM74; HMOXI; HUMCYT2A; ICEBERG; ICOSL; 1D2; IFN-α;IFNA1; IFNA2; IFNA4; IFNA5; IFNA6; IFNA7; IFNB1; IFNgamma; DFNW1; IGBP1;IGF1; IGF1R; IGF2; IGFBP2; IGFBP3; IGFBP6; IL-1; IL10; IL10RA; IL10RB;IL11; IL11RA; IL-12; IL12A; IL12B; IL12RB1; IL12RB2; IL13; IL13RA1;IL13RA2; IL14; IL15; IL15RA; IL16; IL17; IL17B; IL17C; L17R; IL18;IL18BP; IL18R1; IL18RAP; IL19; ILlA; IL1B; ILIF10; IL1F5; IL1F6; IL1F7;IL1F8; IL1F9; IL1HY1; IL1R1; IL1R2; IL1RAP; IL1RAPL1; IL1RAPL2; IL1RL1;IL1RL2, ILIRN; IL2; IL20; IL20Ra; IL21R; IL22; IL-22c; IL22R; IL22RA2;IL23; IL24; IL25; IL26; IL27; IL28A; IL28B; IL29; IL2RA; IL2RB; IL2RG;IL3; IL30; IL3RA; IL4; IL4R; ILS; IL5RA; IL6; IL6R; IL6ST (glycoprotein130); influenza A; influenza B; EL7; EL7R; EL8; IL8RA; DL8RB; IL8RB;DL9; DL9R; DLK; INHA; INHBA; INSL3; INSL4; IRAK1; IRTA2 (Immunoglobulinsuperfamily receptor translocation associated 2); ERAK2; ITGA1; ITGA2;ITGA3; ITGA6 (a6 integrin); ITGAV; ITGB3; ITGB4 (b4 integrin); a407 andaEP7 integrin heterodimers; JAG1; JAK1; JAK3; JUN; K6HF; KAIl; KDR;KITLG; KLF5 (GC Box BP); KLF6; KLKIO; KLK12; KLK13; KLK14; KLK15; KLK3;KLK4; KLK5; KLK6; KLK9; KRT1; KRT19 (Keratin 19); KRT2A; KHTHB6(hair-specific type H keratin); LAMAS; LEP (leptin); LGR5 (leucine-richrepeat-containing G protein-coupled receptor κ; GPR49, GPR67);Lingo-p75; Lingo-Troy; LPS; LTA (TNF-b); LTB; LTB4R (GPR16); LTB4R2;LTBR; LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein ofthe leucine rich repeat (LRR) family); Ly6E (lymphocyte antigen 6complex, locus E; Ly67,RIG-E,SCA-2,TSA-1); Ly6G6D (lymphocyte antigen 6complex, locus G6D; Ly6-D, MEGT1); LY6K (lymphocyte antigen 6 complex,locus K; LY6K; HSJ001348; FLJ35226); MACMARCKS; MAG or OMgp; MAP2K7(c-Jun); MDK; MDP; MIB1; midkine; MEF; MIP-2; MKI67; (Ki-67); MMP2;MMP9; MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor,mesothelin); MS4A1; MSG783 (RNF124, hypothetical protein FLJ20315);MSMB; MT3 (metallothionectin-111); MTSS1; MUC1 (mucin); MYC; MY088;Napi3b (also known as NaPi2b) (NAPI-3B, NPTIIb, SLC34A2, solute carrierfamily 34 (sodium phosphate), member 2, type II sodium-dependentphosphate transporter 3b); NCA; NCK2; neurocan; NFKB1; NFKB2; NGFB(NGF); NGFR; NgR-Lingo; NgR-Nogo66 (Nogo); NgR-p75; NgR-Troy; NME1(NM23A); NOX5; NPPB; NROB1; NROB2; NR1D1; NR1D2; NR1H2; NR1H3; NR1H4;NR112; NR113; NR2C1; NR2C2; NR2E1; NR2E3; NR2F1; NR2F2; NR2F6; NR3C1;NR3C2; NR4A1; NR4A2; NR4A3; NR5A1; NR5A2; NR6A1; NRP1; NRP2; NT5E; NTN4;ODZI; OPRD1; OX40; P2RX7; P2X5 (Purinergic receptor P2X ligand-gated ionchannel 5); PAP; PARTi; PATE; PAWR; PCA3; PCNA; PD-Li; PD-L2; PD-1;POGFA; POGFB; PECAMI; PF4 (CXCL4); PGF; PGR; phosphacan; PIAS2; PIK3CG;PLAU (uPA); PLG; PLXDCI; PMEL17 (silver homolog; SILV; D12S53E; PMEL17;SI; SIL); PPBP (CXCL7); PPID; PRI; PRKCQ; PRKDI; PRL; PROC; PROK2; PSAP;PSCA hlg (2700050C12Rik, C530008016Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene); PTAFR; PTEN; PTGS2 (COX-2); PTN; RAC2 (p21 Rac2);RARB; RET (ret proto-oncogene; MEN2A; HSCR1; MEN2B; MTC1; PTC; CDHF12;Hs.168114; RET51; RET-ELE1); RGSI; RGS13; RGS3; RNF110 (ZNF144); ROBO2;S100A2; SCGB1D2 (lipophilin B); SCGB2A1 (mammaglobin2); SCGB2A2(mammaglobin 1); SCYEI (endothelial Monocyte-activating cytokine); SDF2;Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B); SERPINAI; SERPINA3; SERPINBS (maspin);SERPINEI(PAI-1); SERPDMFI; SHBG; SLA2; SLC2A2; SLC33A1; SLC43A1; SLIT2;SPPI; SPRRIB (Sprl); ST6GAL1; STABI; STAT6; STEAP (six transmembraneepithelial antigen of prostate); STEAP2 (HGNC_8639, IPCA-1, PCANAP1,STAMP1, STEAP2, STMP, prostate cancer associated gene 1, prostate cancerassociated protein 1, six transmembrane epithelial antigen of prostate2, six transmembrane prostate protein); TB4R2; TBX21; TCPIO; TOGFI; TEK;TENB2 (putative transmembrane proteoglycan); TGFA; TGFBI; TGFBlII;TGFB2; TGFB3; TGFBI; TGFBRI; TGFBR2; TGFBR3; THIL; THBSI(thrombospondin-1); THBS2; THBS4; THPO; TIE (Tie-1); TMP3; tissuefactor; TLR1; TLR2; TLR3; TLR4; TLRS; TLR6; TLR7; TLR8; TLR9; TLR10;TMEFF1 (transmembrane protein with EGF-like and two follistatin-likedomains 1; Tomoregulin-1); TMEM46 (shisa homolog 2); TNF; TNF-a; TNFAEP2(B94); TNFAIP3; TNFRSFIIA; TNFRSFIA; TNFRSFIB; TNFRSF21; TNFRSF; TNFRSF6(Fas); TNFRSF7; TNFRSF8; TNFRSF9; TNFSF10 (TRAIL); TNFSF11 (TRANCE);TNFSF12 (APO3L); TNFSF13 (April); TNFSF13B; TNFSF14 (HVEM-L); TNFSF15(VEGI); TNFSFI8; TNFSF4 (OX40 ligand); TNFSF5 (CD40 ligand); TNFSF6(FasL); TNFSF7 (CD27ligand); TNFSFS (CD30ligand); TNFSF9 (4-1 BBligand); TOLLIP; Toll-like receptors; TOP2A (topoisomerase Ea); TP53;TPM1; TPM2; TRADD; TMEM118 (ring finger protein, transmembrane 2; RNFT2;FLJ14627); TRAF1; TRAF2; TRAF3; TRAF4; TRAF5; TRAF6; TREM1; TREM2; TrpM4(BR22450, FLJ20041, TRPM4, TRPM4B, transient receptor potential cationchannel, subfamily M, member 4); TRPC6; TSLP; TWEAK; Tyrosinase (TYR;OCAIA; OCAlA; tyrosinase; SHEP3); VEGF; VEGFB; VEGFC; versican; VHL C5;VLA-4; XCL1 (lymphotactin); XCL2 (SCM-lb); XCRI(GPR5/CCXCRI); YY1;and/or ZFPM2.

In certain embodiments, target molecules for antibodies (or bispecificantibodies) produced according to the methods disclosed herein includeCD proteins such as CD3, CD4, CDS, CD16, CD19, CD20, CD21 (CR2(Complement receptor 2) or C3DR (C3d/Epstein Barr virus receptor) orHs.73792); CD33; CD34; CD64; CD72 (B-cell differentiation antigen CD72,Lyb-2); CD79b (CD79B, CD790, IGb (immunoglobulin-associated beta), B29);CD200 members of the ErbB receptor family such as the EGF receptor,HER2, HER3, or HER4 receptor; cell adhesion molecules such as LFA-1,Mac1, p150.95, VLA-4, ICAM-1, VCAM, alpha4/beta7 integrin, andalphav/beta3 integrin including either alpha or beta subunits thereof(e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies); growth factorssuch as VEGF-A, VEGF-C; tissue factor (TF); alpha interferon (alphaIFN);TNFalpha, an interleukin, such as IL-1 beta, IL-3, IL-4, IL-5, IL-6,IL-8, IL-9, IL-13, IL 17 AF, IL-1S, IL-13R alphal, IL13R alpha2, IL-4R,IL-5R, IL-9R, IgE; blood group antigens; flk2/flt3 receptor; obesity(OB) receptor; mpl receptor; CTLA-4; RANKL, RANK, RSV F protein, proteinC etc. In certain embodiments, the methods provided herein can be usedto produce an antibody (or a multispecific antibody, such as abispecific antibody) that specifically binds to complement protein C5(e.g., an anti-C5 agonist antibody that specifically binds to human C5).

In certain embodiments, the methods provided herein can be used toproduce an antibody (or a multispecific antibody, such as a bispecificantibody) that specifically binds to influenza virus B hemagglutinin,i.e., “fluB” (e.g., an antibody that binds hemagglutinin from theYamagata lineage of influenza B viruses, binds hemagglutinin from theVictoria lineage of influenza B viruses, binds hemagglutinin fromancestral lineages of influenza B virus, or binds hemagglutinin from theYamagata lineage, the Victoria lineage, and ancestral lineages ofinfluenza B virus, in vitro and/or in vivo). Further details regardinganti-FluB antibodies are described in WO 2015/148806, which isincorporated herein by reference in its entirety.

In certain embodiments, an antibody (or bispecific antibody) producedaccording to a method provided herein binds low density lipoproteinreceptor-related protein (LRP)-1 or LRP-8 or transferrin receptor, andat least one target selected from the group consisting of beta-secretase(BACE1 or BACE2), alpha-secretase, gamma-secretase, tau-secretase,amyloid precursor protein (APP), death receptor 6 (DR6), amyloid betapeptide, alpha-synuclein, Parkin, Huntingtin, p75 NTR, CD40 andcaspase-6.

In certain embodiments, the antibody produced according to a methodprovided herein is a human IgG2 antibody against CD40. In certainembodiments, the anti-CD40 antibody is RG7876.

In certain embodiments, the polypeptide produced according to a methodprovided herein is a targeted immunocytokine. In certain embodiments,the targeted immunocytokine is a CEA-IL2v immuocytokine. In certainembodiments, the CEA-IL2v immuocytokine is RG7813. In certainembodiments, the targeted immunocytokine is a FAP-IL2v immuocytokine. Incertain embodiments, the FAP-IL2v immunocytokine is RG7461.

In certain embodiments, a multispecific antibody (such as a bispecificantibody) produced according to a method provided herein binds CEA andat least one additional target molecule. In certain embodiments, amultispecific antibody (such as a bispecific antibody) producedaccording to a method provided herein binds a tumor targeted cytokineand at least one additional target molecule. In certain embodiments, amultispecific antibody (such as a bispecific antibody) producedaccording to a method provided herein is fused to IL2v (i.e., aninterleukin 2 variant) and binds an WI1-based immunocytokine and atleast one additional target molecule. In certain embodiments, amultispecific antibody (such as a bispecific antibody) producedaccording to a method provided herein is a T-cell bispecific antibody(i.e., a bispecific T-cell engager or BiTE).

In certain embodiments, a multispecific antibody (such as a bispecificantibody) produced according to a method provided herein binds to atleast two target molecules selected from: IL-1 alpha and IL-1 beta,IL-12 and IL-1S; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-5and IL-4; IL-13 and IL-1beta; IL-13 and IL-25; IL-13 and TARC; IL-13 andMDC; IL-13 and MEF; IL-13 and TGF-˜; IL-13 and LHR agonist; IL-12 andTWEAK, IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; IL-13 andADAMS, IL-13 and PED2, IL17A and IL17F, CEA and CD3, CD3 and CD19, CD138and CD20; CD138 and CD40; CD19 and CD20; CD20 and CD3; CD3S and CD13S;CD3S and CD20; CD3S and CD40; CD40 and CD20; CD-S and IL-6; CD20 andBR3, TNF alpha and TGF-beta, TNF alpha and IL-1 beta; TNF alpha andIL-2, TNF alpha and IL-3, TNF alpha and IL-4, TNF alpha and IL-5, TNFalpha and IL6, TNF alpha and IL8, TNF alpha and IL-9, TNF alpha andIL-10, TNF alpha and IL-11, TNF alpha and IL-12, TNF alpha and IL-13,TNF alpha and IL-14, TNF alpha and IL-15, TNF alpha and IL-16, TNF alphaand IL-17, TNF alpha and IL-18, TNF alpha and IL-19, TNF alpha andIL-20, TNF alpha and IL-23, TNF alpha and IFN alpha, TNF alpha and CD4,TNF alpha and VEGF, TNF alpha and MIF, TNF alpha and ICAM-1, TNF alphaand PGE4, TNF alpha and PEG2, TNF alpha and RANK ligand, TNF alpha andTe38, TNF alpha and BAFF,TNF alpha and CD22, TNF alpha and CTLA-4, TNFalpha and GP130, TNF a and IL-12p40, VEGF and Angiopoietin, VEGF andHER2, VEGF-A and HER2, VEGF-A and PDGF, HER1 and HER2, VEGFA andANG2,VEGF-A and VEGF-C, VEGF-C and VEGF-D, HER2 and DR5,VEGF and IL-8,VEGF and MET, VEGFR and MET receptor, EGFR and MET, VEGFR and EGFR, HER2and CD64, HER2 and CD3, HER2 and CD16, HER2 and HER3; EGFR (HER1) andHER2, EGFR and HER3, EGFR and HER4, IL-14 and IL-13, IL-13 and CD40L,IL4 and CD40L, TNFR1 and IL-1R, TNFR1 and IL-6R and TNFR1 and IL-18R,EpCAM and CD3, MAPG and CD28, EGFR and CD64, CSPGs and RGM A; CTLA-4 andBTN02; IGF1 and IGF2; IGF1/2 and Erb2B; MAG and RGM A; NgR and RGM A;NogoA and RGM A; OMGp and RGM A; POL-1 and CTLA-4; and RGM A and RGM B.

In certain embodiments, the multispecific antibody (such as a bispecificantibody) is an anti-CEA/anti-CD3 bispecific antibody. In certainembodiments, the anti-CEA/anti-CD3 bispecific antibody is RG7802.Further details regarding anti-CEA/anti-CD3 bispecific antibodies areprovided in WO 2014/121712, which is incorporated herein by reference inits entirety.

In certain embodiments, the multispecific antibody (such as a bispecificantibody) is an anti-VEGF/anti-angiopoietin bispecific antibody. Incertain embodiments, the anti-VEGF/anti-angiopoietin bispecific antibodybispecific antibody is a Crossmab. In certain embodiments, theanti-VEGF/anti-angiopoietin bispecific antibody is RG7716.

In certain embodiments, the multispecific antibody (such as a bispecificantibody) is an anti-Ang2/anti-VEGF bispecific antibody. In certainembodiments, the anti-Ang2/anti-VEGF bispecific antibody is RG7221. Incertain embodiments, the anti-Ang2/anti-VEGF bispecific antibody is CASNumber 1448221-05-3.

Many other antibodies and/or other proteins may be expressed by the hostcells in accordance with the present disclosure, and the above lists arenot meant to be limiting.

The host cells of the present disclosure may be employed in theproduction of a molecule of interest at manufacturing scale.“Manufacturing scale” production of therapeutic proteins, or otherproteins, utilize cell cultures ranging from about 400 L to about 80,000L, depending on the protein being produced and the need. Typically, suchmanufacturing scale production utilizes cell culture sizes from about400 L to about 25,000 L. Within this range, specific cell culture sizessuch as 4,000 L, about 6,000 L, about 8,000, about 10,000, about 12,000L, about 14,000 L, or about 16,000 L may be utilized.

The host cells of the present disclosure can be employed in theproduction of large quantities of a molecule of interest in a shortertimeframe as compared to non-TI cells used in current cell culturemethods. In certain embodiments, the host cells of the presentdisclosure can be employed for improved quality of the molecule ofinterest as compared to non-TI cells used in current cell culturemethods. In certain embodiments, the host cells of the presentdisclosure can be used to enhance seed train stability by preventingchronic toxicity that can be caused by products that can cause cellstress and clonal instability over time. In certain embodiments, thehost cells of the present disclosure can be used for the optimalexpression of acutely toxic products.

In certain embodiments, the host cells, the TI systems of the presentdisclosure, can be used for cell culture process optimization and/orprocess development.

In certain embodiments, the host cells of the present disclosure can beused to accelerate the production of a molecule of interest by about 1week, about, 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeksas compared to non-TI cells used in conventional cell culture methods.In certain embodiments, the host cells of the present disclosure can beused to accelerate the harvest of a molecule of interest by about 1week, about, 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, or about 10 weeksas compared to non-TI cells used in conventional cell culture methods.

In certain embodiments, the host cells of the present embodiment can beemployed to reduce aggregate levels of a molecule of interest ascompared to non-TI cells used in conventional cell culture methods.

In certain embodiments, the host cells of the present disclosure can beused to achieve increased expression of a polypeptide (or polypeptides)of interest relative to a randomly integrated host cell. For example,but not by way of limitation, the host cells of the present disclosurecan achieve expression of standard and half antibodies at titers of atleast 3 g/L, 3.5 g/L, 4 g/L, 4.5 g/L, 5 g/L, 5.5 g/L, 6 g/L, 6.5 g/L, 7g/L, 7.5 g/L, 8 g/L, 8.5 g/L, 9 g/L, 9.5 g/L, 10 g/L, 10.5 g/L, 11 g/L,or more, and expression of multispecific antibodies, e.g., bispecificantibodies, of at least 1.5 g/L, 2 g/L, 2.5 g/L, 3 g/L, 3.5 g/L, 4 g/L,4.5 g/L, 5 g/L, 5.5 g/L, 6 g/L, or more. In certain embodiments, thehost cells of the present disclosure can achieve increased bispecificcontent relative to random integration host cells. For example, but notby way of limitation the host cells of the present disclosure canachieve bispecific content of at least 80%, 85%, 90%, 95%, 96%, 98%, 99%or more.

In certain embodiments, the host cells of the present disclosure can beused for the constitutive expression of selected subunits of atherapeutic molecule and the regulated expression of other, differentsubunits of the same therapeutic molecule. In certain embodiments thetherapeutic molecule can be a fusion protein. In certain embodiments,the host cells of the present disclosure can be used to understand theroles and effects of each antibody subunit in the expression andsecretion of fully assembled antibody molecules.

In certain embodiments, the host cells of the present disclosure can beused as an investigational tool. In certain embodiments, the host cellsof the present disclosure can be used as a diagnostic tool to map outthe root causes of low protein expression for problematic molecules invarious cells. In certain embodiments, the host cells of the presentdisclosure can be used to directly link an observed phenomenon orcellular behavior to the transgene expression in the cells. The hostcell of the present disclosure can also be used to demonstrate whetheror not an observed behavior is reversible in the cells. In certainembodiments, the host cells of the present disclosure can be exploitedto identify and mitigate problems with respect to transgene(s)transcription and expression in cells.

In certain embodiments, the host cells of the present disclosure can beused for swapping transgene subunits, such as but not limited to, HC andLC subunits of an antibody, of a difficult-to-express molecule with thatof an average molecule in the TI system to identify the problematicsubunit(s). In certain embodiments, amino acid sequence analysis canthen be used to narrow down and focus on the amino acid residues orregions that might be responsible for low protein expression.

8. Exemplary Non-Limiting Embodiments

A. A targeted integration (TI) host cell comprising an exogenousnucleotide sequence integrated at an integration site within a specificlocus of the genome of the host cell, wherein the locus is at leastabout 90% homologous to a sequence selected from SEQ ID Nos. 1-7.

A1. The TI host cell of A, wherein the nucleotide sequence immediately5′ of the integrated exogenous nucleotide sequence is selected from thegroup consisting of sequences at least about 90% homologous tonucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 ofNW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides69303-79768 of NW_003616412.1, nucleotides 293481-315265 ofNW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, ornucleotides 82214-97705 of NW_003615411.1.

A2. The TI host cell of A wherein the nucleotide sequence immediately 5′of the integrated exogenous nucleotide sequence is selected from thegroup consisting of sequences at least 15 base pairs, at least 20 basepairs, at least 30 base pairs, at least 40 base pairs, at least 50 basepairs, at least 75 base pairs, at least 100 base pairs, at least 150base pairs, at least 200 base pairs, at least 300 base pairs, at least400 base pairs, at least 500 base pairs, at least 1,000 base pairs, atleast 1,500 base pairs, at least 2,000 base pairs, at least 3,000 basepairs from nucleotide 45269 of NW_006874047.1, nucleotide 207911 ofNW_006884592.1, nucleotide 491909 of NW_006881296.1, nucleotide 79768 ofNW_003616412.1, nucleotide 315265 of NW_003615063.1, nucleotide 2662054of NW_006882936.1, or nucleotide 97705 of NW_003615411.1.

A3. The TI host cell of A, wherein the nucleotide sequence immediately3′ of the integrated exogenous nucleotide sequence is selected from thegroup consisting of sequences at least about 90% homologous tonucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 ofNW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides79769-100059 of NW_003616412.1, nucleotides 315266-362442 ofNW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, ornucleotides 97706-105117 of NW_003615411.1.

A4. The TI host cell of A wherein the nucleotide sequence immediately 3′of the integrated exogenous nucleotide sequence is selected from thegroup consisting of sequences at least 15 base pairs, at least 20 basepairs, at least 30 base pairs, at least 40 base pairs, at least 50 basepairs, at least 75 base pairs, at least 100 base pairs, at least 150base pairs, at least 200 base pairs, at least 300 base pairs, at least400 base pairs, at least 500 base pairs, at least 1,000 base pairs, atleast 1,500 base pairs, at least 2,000 base pairs, at least 3,000 basepairs from nucleotide 45270 of NW_006874047.1, nucleotide 207912 ofNW_006884592.1, nucleotide 491910 of NW_006881296.1, nucleotide 79769 ofNW_003616412.1, nucleotide 315266 of NW_003615063.1, nucleotide 2662055of NW_006882936.1, or nucleotide 97706 of NW_003615411.1 A5. The TI hostcell of any one of A-A4, wherein the integrated exogenous nucleotidesequence is operably linked to a nucleotide sequence selected from thegroup consisting of sequences at least about 90% homologous to asequence selected from SEQ ID Nos. 1-7.

A6. A TI host cell comprising an exogenous nucleotide sequenceintegrated at an integration site within an endogenous gene selectedfrom the group consisting of LOC107977062, LOC100768845, ITPR2,ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about90% homologous thereto.

A7. A TI host cell comprising an exogenous nucleotide sequenceintegrated at an integration site operably linked to an endogenous geneselected from the group consisting of LOC107977062, LOC100768845, ITPR2,ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about90% homologous thereto.

A8. A TI host cell comprising an exogenous nucleotide sequenceintegrated at an integration site immediately adjacent to all or aportion of a sequence selected from the group consisting of sequences atleast about 90% homologous to a sequence selected from SEQ ID Nos. 1-7.

A9. The TI host cell of any one of A-A8, wherein the TI host cell is amammalian host cell.

A10. The TI host cell of A9, wherein the TI host cell is a hamster hostcell, a human host cell, a rat host cell, or a mouse host cell.

A11. The TI host cell of A9 or A10, wherein the TI host cell is aChinese hamster ovary (CHO) host cell, a CHO K1 host cell, a CHO KISVhost cell, a DG44 host cell, a DUKXB-I1 host cell, a CHOK1S host cell,or a CHO KIM host cell.

A12. The TI host cell of any one of A-A11, wherein the exogenousnucleotide sequence comprises one or more recombination recognitionsequences (RRSs), wherein the RRS can be recognized by a recombinase.

A13. The TI host cell of A12, wherein the exogenous nucleotide sequencecomprises at least two RRSs.

A14. The TI host cell of A12 or A13, wherein the recombinase is a Crerecombinase or an FLP recombinase.

A15. The TI host cell of A12 or A13, wherein the recombinase is a Bxb1integrase or a φC31 integrase.

A16. The TI host cell of any one of A12-A15, wherein the RRS is selectedfrom the group consisting of a LoxP sequence, a LoxP L3 sequence, a LoxP2 L sequence, a LoxFas sequence, a Lox511 sequence, a Lox2272 sequence,a Lox2372 sequence, a Lox5171 sequence, a Loxm2 sequence, a Lox71sequence, a Lox66 sequence, a FRT sequence, a Bxb1 attP sequence, a Bxb1attB sequence, a pC31 attP sequence, and a pC31 attB sequence.

A17. The TI host cell of any one of A13-A16, wherein the exogenousnucleotide sequence comprises a first and a second RRS, and at least oneselection marker located between the first and the second RRS.

A18. The TI host cell of A17, comprising a first selection marker,wherein the first selection marker is selected from the group consistingof aminoglycoside phosphotransferase (APH), hygromycinphosphotransferase (HYG), neomycin, G418 APH), dihydrofolate reductase(DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparaginesynthetase, tryptophan synthetase (indole), histidinol dehydrogenase(histidinol D), blasticidin, bleomycin, phleomycin, chloramphenicol,Zeocin, and mycophenolic acid.

A19. The TI host cell of A17 or A18, further comprising a secondselection marker, wherein the first and the second selection markers aredifferent.

A20. The TI host cell of A19, wherein the second selection marker isselected from the group consisting of aminoglycoside phosphotransferase(APH), hygromycin phosphotransferase (HYG), neomycin, G418 APH),dihydrofolate reductase (DHFR), thymidine kinase (TK), glutaminesynthetase (GS), asparagine synthetase, tryptophan synthetase (indole),histidinol dehydrogenase (histidinol D), blasticidin, bleomycin,phleomycin, chloramphenicol, Zeocin, and mycophenolic acid.

A21. The TI host cell of A19 or A20, further comprising a thirdselection marker and an internal ribosome entry site (IRES), wherein theIRES is operably linked to the third selection marker.

A22. The TI host cell of A21, wherein the third selection marker isdifferent from the first or the second selection marker.

A23. The TI host cell of A21 or A22, wherein the third selection markeris selected from the group consisting of a green fluorescent protein(GFP) marker, an enhanced GFP (eGFP) marker, a synthetic GFP marker, ayellow fluorescent protein (YFP) marker, an enhanced YFP (eYFP) marker,a cyan fluorescent protein (CFP) marker, a mPlum marker, a mCherrymarker, a tdTomato marker, a mStrawberry marker, a J-red marker, aDsRed-monomer marker, a mOrange marker, a mKO marker, a mCitrine marker,a Venus marker, a YPet marker, an Emerald6 marker, a CyPet marker, amCFPm marker, a Cerulean marker, and a T-Sapphire marker.

A24. The TI host cell of anyone of A17-A23, further comprising a thirdRRS, wherein the third RRS is located between the first and the secondRRS, and the third RRS is heterospecific relative the first or thesecond RRS.

A25. The TI host cell of any one of A12-A24, wherein the exogenousnucleotide sequence comprises at least one selection marker and at leastone exogenous sequence of interest (SOI).

A26. The TI host cell any one of A12-A25, wherein the exogenousnucleotide sequence further comprises at least one exogenous SOI.

A27. The TI host cell of A26, wherein the exogenous SOI is locatedbetween the first and the second RRS.

A28. The TI host cell of A26 or A27, wherein the SOI encodes a singlechain antibody, an antibody light chain, an antibody heavy chain, asingle-chain Fv fragment (scFv), or an Fc fusion protein.

A29. The TI host cell of any one of A24-A28, wherein the exogenousnucleotide sequence further comprises at least one exogenous SOI locatedbetween the first and the third RRS, and at least one exogenous SOIlocated between the third and the second RRS.

A30. The TI host cell of A29, wherein the SOI encodes a single chainantibody, an antibody light chain, an antibody heavy chain, asingle-chain Fv fragment (scFv), or an Fc fusion protein.

A31. The TI host cell of A26 wherein the SOI encodes an antibodyexpressed at a higher level relative to a randomly integrated host cell.

A32. The TI host cell of A31 where the antibody is a standard or halfantibodies expressed at a titer of at least 3 g/L, 3.5 g/L, 4 g/L, 4.5g/L, 5 g/L, 5.5 g/L, 6 g/L, 6.5 g/L, 7 g/L, 7.5 g/L, 8 g/L, 8.5 g/L, 9g/L, 9.5 g/L, 10 g/L, 10.5 g/L, 11 g/L, or more,

A33. The TI host cell of A31 where the antibody is a multispecificantibody, e.g., a bispecific antibody, expressed at a titer of at least1.5 g/L, 2 g/L, 2.5 g/L, 3 g/L, 3.5 g/L, 4 g/L, 4.5 g/L, 5 g/L, 5.5 g/L,6 g/L.

A34. The TI host cell of A31 where the antibody is a bispecific antibodyand the bispecific content is increased relative to a random integrationhost cell.

A35. The TI host cell of A1 wherein the antibody is a bispecificantibody and the bispecific content is at least 80%, 85%, 90%, 95%, 96%,98%, 99% or more.

B. A method of preparing a TI host cell expressing a polypeptide ofinterest comprising: providing a TI host cell comprising an exogenousnucleotide sequence integrated at a site within a locus of the genome ofthe TI host cell, wherein the locus is at least about 90% homologous toa sequence selected from SEQ ID No. 1-7, wherein the exogenousnucleotide sequence comprises two RRSs, flanking at least one firstselection marker; introducing into the cell provided in a) a vectorcomprising two RRSs matching the two RRSs on the integrated exogenousnucleotide sequence and flanking at least one exogenous SOI and at leastone second selection marker; introducing a recombinase or a nucleic acidencoding a recombinase, wherein the recombinase recognizes the RRSs; andselecting for TI cells expressing the second selection marker to therebyisolate a TI host cell expressing the polypeptide.

B1. The method of B, wherein the recombinase is Cre recombinase or anFLP recombinase.

B2. The method of B, wherein the recombinase is a Bxb integrase or apC31 integrase.

B3. The method of any one of B-B2, wherein the SOI encodes a singlechain antibody, an antibody light chain, an antibody heavy chain, asingle-chain Fv fragment (scFv), or an Fc fusion protein.

B4. The method of any one of claims B-B3, wherein the TI host cell is amammalian host cell.

B5. The method of B4, wherein the TI host cell is a hamster host cell, ahuman host cell, a rat host cell, or a mouse host cell.

B6. The method of B4 or B5, wherein the TI host cell is a CHO host cell,a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11host cell, a CHOK1S host cell, or a CHO KIM host cell.

C. A method for expressing a polypeptide of interest comprising:providing a TI host cell comprising at least one exogenous SOI and atleast one selection marker where the at least one exogenous SOI and atleast one selection marker are flanked by two RRSs integrated within alocus of the genome of the TI host cell, wherein the locus is at leastabout 90% homologous to a sequence selected from SEQ ID Nos. 1-7; andculturing the cell in a) under conditions suitable for expressing thepolypeptide of interest and recovering a polypeptide of interesttherefrom.

C1. The method of C, wherein the at least one exogenous SOI encodes asingle chain antibody, an antibody light chain, an antibody heavy chain,a single-chain Fv fragment (scFv), or an Fc fusion protein.

D. A method for preparing a TI host cell expressing at least a first andsecond polypeptide of interest comprising: providing a TI host cellcomprising an exogenous nucleotide sequence integrated at a site withina locus of the genome of the host cell, wherein the locus is at leastabout 90% homologous to a sequence selected from SEQ ID Nos. 1-7,wherein the exogenous nucleotide sequence comprises a first and a secondRRS flanking at least one first selection marker, and a third RRSlocated between the first and the second RRS, and all the RRSs areheterospecific; introducing into the cell provided in a) a first vectorcomprising two RRSs matching the first and the third RRS on theintegrated exogenous nucleotide sequence and flanking at least one firstexogenous SOI and at least one second selection marker; introducing intothe cell provided in a) a second vector comprising two RRSs matching thesecond and the third RRS on the integrated exogenous nucleotide sequenceand flanking at least one second exogenous SOI; introducing one or morerecombinases, or one or more nucleic acids encoding one or morerecombinases, wherein the one or more recombinases recognize the RRSs;and selecting for TI cells expressing the second selection marker tothereby isolate a TI host cell expressing the first and secondpolypeptides of interest.

D1. The method of D, wherein the recombinase is Cre recombinase or anFLP recombinase.

D2. The method of D, wherein the recombinase is a Bxb1 integrase or a(pC31 integrase.

D3. The method of D, wherein: the first vector further comprises apromoter sequence operably linked to the codon ATG positioned flankedupstream by the first SOI and downstream by an RRS; and the secondvector further comprises a selection marker lacking an ATG transcriptionstart codon flanked upstream by an RRS and downstream by the second SOI.

D4. The method of any one of D-D3, wherein the first SOI encodes asingle chain antibody, an antibody light chain, an antibody heavy chain,a single-chain Fv fragment (scFv), or an Fc fusion protein.

D5. The method of any one of D-D3, wherein the second SOI encodes asingle chain antibody, an antibody light chain, an antibody heavy chain,a single-chain Fv fragment (scFv), or an Fc fusion protein.

D6. The method of any one of C or D-D5, wherein the TI host cell is amammalian host cell.

D7. The method of D6, wherein the TI host cell is a hamster host cell, ahuman host cell, a rat host cell, or a mouse host cell.

D8. The method of D7, wherein the TI host cell is a CHO host cell, a CHOK1 host cell, a CHO K1 SV host cell, a DG44 host cell, a DUKXB-11 hostcell, a CHOK1S host cell, or a CHO K1M host cell.

E. A method for expressing a polypeptide of interest comprising:providing a host cell comprising at least two exogenous SOIs and atleast one selection marker integrated within a locus of the genome ofthe host cell, wherein the locus is at least about 90% homologous to asequence selected from SEQ ID Nos 1-7, wherein at least one exogenousSOI and one selection marker is flanked by a first and a third RRS andat least one exogenous SOI is flanked by a second and the third RRS; andculturing the cell in a) under conditions suitable for expressing thepolypeptide of interest and recovering a polypeptide of interesttherefrom.

E1. The method of E, wherein the first SOI encodes a single chainantibody, an antibody light chain, an antibody heavy chain, asingle-chain Fv fragment (scFv), or an Fc fusion protein.

E2. The method of E or E1, wherein the second SOI encodes a single chainantibody, an antibody light chain, an antibody heavy chain, asingle-chain Fv fragment (scFv), or an Fc fusion protein.

E3. The method of any one of E-E2, wherein the TI host cell is amammalian host cell.

E4. The method of E3, wherein the TI host cell is a hamster host cell, ahuman host cell, a rat host cell, or a mouse host cell.

E5. The method of E4, wherein the TI host cell is a CHO host cell, a CHOK1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-11 hostcell, a CHOK1S host cell, or a CHO KIM host cell.

F. A vector comprising two nucleotide sequences at least 50% homologousto

a) two reference sequences selected from any portion of SEQ ID No. 1;

b) two reference sequences selected from any portion of SEQ ID No. 2;

c) two reference sequences selected from any portion of SEQ ID No. 3;

d) two reference sequences selected from any portion of SEQ ID No. 4;

e) two reference sequences selected from any portion of SEQ ID No 5;

f) two reference sequences selected from any portion of SEQ ID No. 6; or

g) two reference sequences selected from any portion of SEQ ID No. 7,wherein said sequences flank a DNA cassette, wherein the DNA cassettecomprises at least one selection marker and at least one exogenous SOIflanked by two RRSs.

F1. The vector of F, wherein the vector comprises two nucleotidesequences at least 60% homologous to the two reference sequences.

F2. The vector of F, wherein the vector comprises two nucleotidesequences at least 70% homologous to the two reference sequences.

F3. The vector of F, wherein the vector comprises two nucleotidesequences at least 80% homologous to the two reference sequences.

F4. The vector of F, wherein the vector comprises two nucleotidesequences at least 90% homologous to the two reference sequences.

F5. The vector of F, wherein the vector comprises two nucleotidesequences at least 95% homologous to the two reference sequences.

F6. The vector of F, wherein the vector comprises two nucleotidesequences at least 99% homologous to the two reference sequences.

F7. The vector of any of F-F6, wherein the vector is selected from thegroup consisting of an adenovirus vector, an adeno-associated virusvector, a lentivirus vector, a retrovirus vector, an integrating phagevector, a non-viral vector, a transposon and/or transposase vector, anintegrase substrate, and a plasmid.

F8. The vector of any of F-F7, wherein the SOI encodes a single chainantibody, an antibody light chain, an antibody heavy chain, asingle-chain Fv fragment (scFv), or an Fc fusion protein.

G. A method for preparing a TI host cell expressing a polypeptide ofinterest comprising: providing a host cell comprising a locus of thegenome of the host cell, wherein the locus is at least about 90%homologous to a sequence selected from SEQ ID Nos. 1-7; introducing avector into the host cell, wherein the vector comprises nucleotidesequences at least 50% homologous to a sequence selected from SEQ IDNos. 1-7 flanking a DNA cassette, wherein the DNA cassette comprises atleast one selection marker and at least one exogenous SOI flanked by twoRRSs; and selecting for the selection marker to isolate a TI host cellwith the SOI integrated in the locus of the genome and expressing thepolypeptide of interest.

G1. The method of G, wherein the vector is selected from the groupconsisting of an adenovirus vector, an adeno-associated virus vector, alentivirus vector, a retrovirus vector, an integrating phage vector, anon-viral vector, a transposon and/or transposase vector, an integrasesubstrate, and a plasmid.

G2. The method of G or G1, wherein the at least one SOI encodes a singlechain antibody, an antibody light chain, an antibody heavy chain, asingle-chain Fv fragment (scFv), or an Fc fusion protein.

G3. The method of any one of G-G2, wherein the TI host cell is amammalian host cell.

G4. The method of G3, wherein the TI host cell is a hamster host cell, ahuman host cell, a rat host cell, or a mouse host cell.

G5. The method of G3 or G4, wherein the TI host cell is a CHO host cell,a CHO K1 host cell, a CHO K1SV host cell, a DG44 host cell, a DUKXB-Ihost cell, a CHOK1S host cell, or a CHO KIM host cell.

G6. The method of any one of G1-G5, wherein the integration of thenucleic acid comprising the at least one SOI and selection marker ispromoted by an exogenous nuclease.

G7. The method of G6, wherein the exogenous nuclease is selected fromthe group consisting of a zinc finger nuclease (ZFN), a ZFN dimer, atranscription activator-like effector nuclease (TALEN), a TAL effectordomain fusion protein, an RNA-guided DNA endonuclease, an engineeredmeganuclease, and a clustered regularly interspaced short palindromicrepeats (CRISPR)-associated (Cas) endonuclease.

G8. The method of G, wherein the polypeptide of interest is amultispecific antibody.

G9. The method of G8, wherein the multispecific antibody is a bispecificantibody.

H. A TI host cell comprising at least one exogenous nucleotide sequenceintegrated at an integration site within one or more specific loci ofthe genome of the host cell, wherein the locus is at least about 90%homologous to a sequence selected from SEQ ID Nos. 1-7.

H1. A TI host cell comprising at least one exogenous nucleotide sequenceintegrated at one or more integration sites within an endogenous geneselected from the group consisting of LOC107977062, LOC100768845, ITPR2,ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about90% homologous thereto.

H2. The TI host cell of any one of H-H1, wherein the at least oneexogenous nucleotide sequence comprises one or more recombinationrecognition sequences (RRSs), wherein the RRS can be recognized by arecombinase.

H3. The TI host cell of H2, wherein the at least one exogenousnucleotide sequence further comprises at least one exogenous SOI.

H4. The TI host cell of any of H-H3, wherein the host cell comprises atleast one exogenous nucleotide sequence at a first locus of the genomeof the host cell and at least one exogenous nucleotide sequence at oneor more secondary locus of the genome of the host cell.

H5. The TI host cell of H4 where the first locus comprises a sequence atleast 90% homologous to all or a portion SEQ ID No. 1 and the secondarylocus comprises at least one sequence at least 90% homologous to all ora portion of SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, or SEQ ID No. 7.

H5.1. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 1 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 2.

H5.2. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 1 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 3.

H5.3. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 1 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 4.

H5.4. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 1 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 5.

H5.5. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 1 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 6.

H5.6. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 1 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 7.

H6. The TI host cell of H4 where the first locus comprises a sequence atleast 90% homologous to all or a portion SEQ ID No. 2 and the secondarylocus comprises at least one sequence at least 90% homologous to all ora portion of SEQ ID No. 1, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, or SEQ ID No. 7.

H6.1. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 2 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 1.

H6.2. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 2 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 3.

H6.3. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 2 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 4.

H6.4. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 2 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 5.

H6.5. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 2 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 6.

H6.6. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 2 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 7.

H7. The TI host cell of H4 where the first locus comprises a sequence atleast 90% homologous to all or a portion SEQ ID No. 3 and the secondarylocus comprises at least one sequence at least 90% homologous to all ora portion of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 4, SEQ ID No. 5, SEQID No. 6, or SEQ ID No. 7.

H7.1. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 3 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 1.

H7.2. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 3 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 2.

H7.3. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 3 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 4.

H7.4. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 3 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 5.

H7.5. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 3 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 6.

H7.6. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 3 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 7.

H8. The TI host cell of H4 where the first locus comprises a sequence atleast 90% homologous to all or a portion SEQ ID No. 4 and the secondarylocus comprises at least one sequence at least 90% homologous to all ora portion of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 5, SEQID No. 6, or SEQ ID No. 7.

H8.1. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 4 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 1.

H8.2. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 4 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 2.

H8.3. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 4 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 3.

H8.4. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 4 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 5.

H8.5. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 4 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 6.

H8.6. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 4 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 7.

H9. The TI host cell of H4 where the first locus comprises a sequence atleast 90% homologous to all or a portion SEQ ID No. 5 and the secondarylocus comprises at least one sequence at least 90% homologous to all ora portion of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQID No. 6, or SEQ ID No. 7.

H9.1. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 5 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 1. H9.2. The TI host cell of H4 wherethe first locus comprises a sequence at least 90% homologous to all or aportion SEQ ID No. 5 and the secondary locus comprises at least onesequence at least 90% homologous to all or a portion of SEQ ID No. 2.

H9.3. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 5 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 3.

H9.4. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 5 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 4.

H9.5. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 5 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 6.

H9.6. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 5 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 7.

H10. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 6 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ IDNo. 4, SEQ ID No. 5, or SEQ ID No. 7.

H10.1. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 6 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 1.

H10.2. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 6 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 2.

H10.3. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 6 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 3.

H10.4. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 6 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 4.

H10.5. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 6 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 5.

H10.6. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 6 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 7.

H11. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 7 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 3, SEQ IDNo. 4, SEQ ID No. 5, or SEQ ID No. 6.

H11.1. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 7 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 1.

H11.2. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 7 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 2.

H11.3. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 7 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 3.

H11.4. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 7 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 4.

H11.5. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 7 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 5.

H11.6. The TI host cell of H4 where the first locus comprises a sequenceat least 90% homologous to all or a portion SEQ ID No. 7 and thesecondary locus comprises at least one sequence at least 90% homologousto all or a portion of SEQ ID No. 6.

H12. The TI host cell of H4 where the first locus is an integrationsites within an LOC107977062 gene and the secondary locus is anintegration site within a gene selected from the following group:LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, andsequences at least about 90% homologous thereto.

H13. The TI host cell of H4 where the first locus is an integrationsites within an LOC100768845 gene and the secondary locus is anintegration site within a gene selected from the following group:LOC107977062, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, andsequences at least about 90% homologous thereto.

H14. The TI host cell of H4 where the first locus is an integrationsites within an ITPR2 gene and the secondary locus is an integrationsite within a gene selected from the following group: LOC107977062,LOC100768845, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences atleast about 90% homologous thereto.

H15. The TI host cell of H4 where the first locus is an integrationsites within an ERE67000.1 gene and the secondary locus is anintegration site within a gene selected from the following group:LOC107977062, LOC100768845, ITPR2, UBAP2, MTMR2, XP_003512331.2, andsequences at least about 90% homologous thereto.

H16. The TI host cell of H4 where the first locus is an integrationsites within an UBAP2 gene and the secondary locus is an integrationsite within a gene selected from the following group: LOC107977062,LOC100768845, ITPR2, ERE67000.1, MTMR2, XP_003512331.2, and sequences atleast about 90% homologous thereto.

H17. The TI host cell of H4 where the first locus is an integrationsites within an MTMR2 gene and the secondary locus is an integrationsite within a gene selected from the following group LOC107977062,LOC100768845, ITPR2, ERE67000.1, UBAP2, XP_003512331.2, and sequences atleast about 90% homologous thereto.

H18. The TI host cell of H4 where the first locus is an integrationsites within an XP_003512331.2 gene and the secondary locus is anintegration site within a gene selected from the following group:LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, andsequences at least about 90% homologous thereto.

H19. The TI host cell of H4-H18 wherein the at least one exogenousnucleotide sequence incorporated at the first locus comprises aregulatable promotor.

H20. The TI host cell of H4-H18, wherein the at least one exogenousnucleotide sequence incorporated at the secondary locus comprises aregulatable promoter.

H21. The TI host cell of H4-H18, where the at least one exogenousnucleotide sequence incorporated at the first locus and the at least onenucleotide sequence incorporated at the secondary locus comprise aregulatable promoter.

J. A method of preparing a TI host cell expressing at least onepolypeptide of interest comprising: providing a TI host cell comprisingat least one exogenous nucleotide sequence integrated at a site withinone or more loci of the genome of the TI host cell, wherein the one ormore loci are at least about 90% homologous to a sequence selected fromSEQ ID No. 1-7, wherein the at least one exogenous nucleotide sequencecomprises two RRSs, flanking at least one first selection marker;introducing into the cell provided in a) a vector comprising two RRSsmatching the two RRSs on the integrated exogenous nucleotide sequenceand flanking at least one exogenous SOI and at least one secondselection marker; introducing a recombinase or a nucleic acid encoding arecombinase, wherein the recombinase recognizes the RRSs; and selectingfor TI cells expressing the second selection marker to thereby isolate aTI host cell expressing the at least one polypeptide of interest.

J1. A method for preparing a TI host cell expressing at least one firstand second polypeptide of interest comprising: providing a TI host cellcomprising at least one exogenous nucleotide sequence integrated at asite within one or more loci of the genome of the host cell, wherein oneor more loci are at least about 90% homologous to a sequence selectedfrom SEQ ID Nos. 1-7, wherein the exogenous nucleotide sequencecomprises a first and a second RRS flanking at least one first selectionmarker, and a third RRS located between the first and the second RRS,and all the RRSs are heterospecific; introducing into the cell providedin a) a first vector comprising two RRSs matching the first and thethird RRS on the at least one integrated exogenous nucleotide sequenceand flanking at least one first exogenous SOI and at least one secondselection marker; introducing into the cell provided in a) a secondvector comprising two RRSs matching the second and the third RRS on theat least one integrated exogenous nucleotide sequence and flanking atleast one second exogenous SOI; introducing one or more recombinases, orone or more nucleic acids encoding one or more recombinases, wherein theone or more recombinases recognize the RRSs; and selecting for TI cellsexpressing the second selection marker to thereby isolate a TI host cellexpressing the at least one first and second polypeptides of interest.

J2. A method of preparing a TI host cell expressing a polypeptide ofinterest comprising: a) providing a TI host cell comprising at least oneexogenous nucleotide sequence integrated at a site within one or moreloci of the genome of the TI host cell, wherein the one or more loci areat least about 90% homologous to a sequence selected from SEQ ID No.1-7, wherein the exogenous nucleotide sequence comprises one or moreRRSs; b) introducing into the cell provided in a) a vector comprisingone or more RRSs matching the one or more RRSs on the integratedexogenous nucleotide sequence and flanking at least one exogenous SOIand operably linked to at least one regulatable promoter; c) introducinga recombinase or a nucleic acid encoding a recombinase, wherein therecombinase recognizes the RRSs; and d) selecting for TI cellsexpressing the exogenous polypeptide of interest in the presence of aninducer to thereby isolate a TI host cell expressing the polypeptide ofinterest.

J3. A method for expressing a polypeptide of interest comprising: a)providing a host cell comprising at least one exogenous SOI flanked bytwo RRSs and a regulatable promoter integrated within a locus of thegenome of the host cell, wherein the locus is at least about 90%homologous to a sequence selected from SEQ ID Nos. 1-7; and b) culturingthe cell under conditions suitable for expressing the SOI and recoveringa polypeptide of interest therefrom.

J4. A method for preparing a TI host cell expressing a first and secondpolypeptide of interest comprising: a) providing a TI host cellcomprising an exogenous nucleotide sequence integrated at a site withina locus of the genome of the host cell, wherein the locus is at leastabout 90% homologous to a sequence selected from SEQ ID Nos. 1-7,wherein the exogenous nucleotide sequence comprises a first, second RRSand a third RRS located between the first and the second RRS, and allthe RRSs are heterospecific; b) introducing into the cell provided in a)a first vector comprising two RRSs matching the first and the third RRSon the integrated exogenous nucleotide sequence and flanking at leastone first exogenous SOI operably linked to a regulatable promoter; c)introducing into the cell provided in a) a second vector comprising twoRRSs matching the second and the third RRS on the integrated exogenousnucleotide sequence and flanking at least one second exogenous SOIoperably linked to a regulatable promoter; d) introducing one or morerecombinases, or one or more nucleic acids encoding one or morerecombinases, wherein the one or more recombinases recognize the RRSs;and e) selecting for TI cells expressing the first and second exogenouspolypeptides of interest in the presence of an inducer to therebyisolate a TI host cell expressing the first and second polypeptides ofinterest.

J5. The method of any of claims J, J2, and J3, wherein the integrationof a nucleic acid comprising at least one SOI is promoted by anexogenous nuclease.

J6. The method of claims J1 or J4, wherein the first SOI encodes asingle chain antibody, an antibody light chain, an antibody heavy chain,a single-chain Fv fragment (scFv), or an Fc fusion protein; and thesecond SOI encodes a single chain antibody, an antibody light chain, anantibody heavy chain, a single-chain Fv fragment (scFv), or an Fc fusionprotein.

J7. The method of J5, wherein the exogenous nuclease is selected fromthe group consisting of a zinc finger nuclease (ZFN), a ZFN dimer, atranscription activator-like effector nuclease (TALEN), a TAL effectordomain fusion protein, an RNA-guided DNA endonuclease, an engineeredmeganuclease, and a clustered regularly interspaced short palindromicrepeats (CRISPR)-associated (Cas) endonuclease.

J8. The method of any of claims J-J4, wherein the regulatable promoteris selected from the group consisting of SV40 and CMV promoters.

J9. The method of any of claims J2 and J4, wherein: the first vectorfurther comprises a promoter sequence operably linked to the codon ATGpositioned flanked upstream by the first SOI and downstream by an RRS;and the second vector further comprises a selection marker lacking anATG transcription start codon flanked upstream by an RRS and downstreamby the second SOI.

K. A vector comprising two nucleotide sequences at least 50% homologousto

a) two reference sequences selected from any portion of SEQ ID No. 1;

b) two reference sequences selected from any portion of SEQ ID No. 2;

c) two reference sequences selected from any portion of SEQ ID No. 3;

d) two reference sequences selected from any portion of SEQ ID No. 4;

e) two reference sequences selected from any portion of SEQ ID No 5;

f) two reference sequences selected from any portion of SEQ ID No. 6; or

g) two reference sequences selected from any portion of SEQ ID No. 7,wherein the two nucleotide sequences flank a DNA cassette, wherein theDNA cassette comprises at least one exogenous SOI operably linked to aregulatable promoter and flanked by two RRSs.

K1. The vector of K, wherein the vector comprises two nucleotidesequences at least 60% homologous to the two reference sequences.

K2. The vector of K, wherein the vector comprises two nucleotidesequences at least 70% homologous to the two reference sequences.

K3. The vector of K, wherein the vector comprises two nucleotidesequences at least 80% homologous to the two reference sequences.

K4. The vector of K, wherein the vector comprises two nucleotidesequences at least 90% homologous to the two reference sequences.

K5. The vector of K, wherein the vector comprises two nucleotidesequences at least 95% homologous to the two reference sequences.

K6. The vector of K, wherein the vector comprises two nucleotidesequences at least 99% homologous to the two reference sequences.

K7. The vector of any of K-K6, wherein the vector is selected from thegroup consisting of an adenovirus vector, an adeno-associated virusvector, a lentivirus vector, a retrovirus vector, an integrating phagevector, a non-viral vector, a transposon and/or transposase vector, anintegrase substrate, and a plasmid.

K8. The vector of any of K-K7, wherein the SOI encodes a single chainantibody, an antibody light chain, an antibody heavy chain, asingle-chain Fv fragment (scFv), or an Fc fusion protein.

EXAMPLES

The following examples are merely illustrative of the presentlydisclosed subject matter and should not be considered as limitations inany way.

Example 1: Discovering Highly Productive Targeted Integration Sites forCHO Host Cells for Clinical and Commercial Cell Line Development

This Example describes the methods for identifying targeted integrationloci in the CHO genome having high productivity. Conventional cell linedevelopment (CLD) relies on the random integration (RI) of the plasmidcarrying the sequence of interest (SOI). This process is unpredictable,and labor intensive. As such, a large effort is required to identify thehigh producing RI clones. Unlike the conventional RI CLD, targetedintegration (TI) CLD introduces the transgene at a predetermined“hot-spot” in the CHO genome with a defined copy number (usually 1-2copies). Given the low copy number and the pretested integration site,TI cell lines should have better stability compared to the RI lines.Moreover, since the selective marker is only used for selecting cellswith proper TI and not for selecting cells with a high level oftransgene expression, a less mutagenic marker may be applied to minimizethe chance of sequence variants (SVs), which is in part due to themutagenicity of the selective agents like methotrexate (MTX) ormethionine sulfoximine (MSX).

FIG. 1 is a diagram showing an outline of the genome-wide screeningsteps for identifying CHO TI loci that allow for stable and highexpression of antibodies. To screen for transcriptionally activesequences in the CHO genome, two approaches were utilized to introducean antibody cassette, expressing either antibody A or antibody B, andwhere the antibody sequences were flanked by RRS1 and RRS2, into the CHOgenome.

One approach was the conventional random integration method, and theother was a transposase-based integration that required cotransfectionof a transposase expressing plasmid and the antibody plasmid. 15 k-20 ktransfectants were screened for each of the methods. Based on an intactIgG ELISA assay and gene copy analysis, a total of ˜300 clones with highantibody titer and low HC gene copy numbers were expanded for fed-batchevaluation in shake flasks. 40 clones, representing 40 potentiallydifferent highly transcriptionally active integration sites identifiedby the two methods, with acceptable titer and product qualityattributes, were then selected for GFP landing pad swapping to generateTI hosts.

To replace the antibody cassette in the transcriptionally active loci, alanding pad that encoded a GFP gene and appropriate selection markers,flanked by the same RRS1 and RRS2, was constructed for RMCE. To initiateRMCE, the recombinase and the GFP landing pad were cotransfected intoeach of the 40 top clones. Two selection markers were used to enrich thetargeted integration events. Successful RMCE should lead to the GFPlanding pad being targeted to the transcriptionally active locus andreplacing the antibody cassette, resulting in gain of GFP expression andloss of the antibody expression. The change in phenotype from GFP−/mAB+to GFP+/mAb− should be readily detectable using FACS analysis. GFP+/mAb−enrichment was detected in 14 out of the 40 RMCE pools using FACSanalysis. To isolate individual TI host candidates, each of these 14RMCE pools was then single cell cloned. A total of 90 potential TI hostcandidates from the 14 pools were selected based on FACS and genomic PCRconfirmation that the original antibody cassette was removed andreplaced by the GFP landing pad at the integration sites of interest.Subsequently, some of these candidate hosts were evaluated for theirRMCE capability and efficiency using a test antibody C flanked by thesame RRS1 and RRS2. Two double selection schemes were utilized togenerate the targeted antibody expressing population (FIGS. 2A and 2B),depending on which method the transcriptionally active locus wasidentified with. The configurations of the landing pads were slightlydifferent for the two screening methods. Based on the RMCE efficiency aswell as antibody productivity of the TI transfection pools, seven finalhosts were selected, representing seven unique integration sites in theCHOK1M hosts for high antibody expression.

These seven hosts were analyzed by Targeted Locus Amplification/NextGeneration Sequencing (TLA/NGS) to identify the CHO genomic sequencesflanking the integration sites, thus providing the potential genesequences the integration sites of interest reside in.

TABLE 2 TI host cell integration sites Contig Integration Host ContigSize (kb) site (bp) Gene (SEQ ID No.) 1 NW_006874047.1 727 45269LOC107977062 (SEQ ID No. 1) 2 NW_006884592.1 931 207911 LOC100768845(SEQ ID No. 2) 3 NW_006881296.1 1016 491909 ITPR2 (SEQ ID No. 3) 4NW_003616412.1 127 79768 ERE67000.1 (SEQ ID No. 4) 5 NW_003615063.1 372315265 UBAP2 (SEQ ID No. 5) 6 NW_006882936.1 3042 2662054 MTMR2 (SEQ IDNo. 6) 7 NW_003615411.1 277 97706 XP_003512331.2 (SEQ ID No. 7)

Based on the integration sites, 5′ flanking sequences can include:nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 ofNW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides69303-79768 of NW_003616412.1, nucleotides 293481-315265 ofNW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, andnucleotides 82214-97705 of NW_003615411.1.

Based on the integration sites, 3′ flanking sequences can include:nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 ofNW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides79769-100059 of NW_003616412.1, nucleotides 315266-362442 ofNW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, andnucleotides 97706-105117 of NW_003615411.1.

For two of the hosts, hosts 4 and 7, further analysis indicated that asingle GFP landing pad was inserted into the CHO host genome. Noantibody sequence was found in these two hosts, indicating the completeremoval of the initial antibody cassettes used to identify thetranscriptionally active loci. In addition, whole genome sequencing wasperformed in house for genomic DNA isolated from these two TI hosts. Noantibody specific sequences were detected in these two TI hosts, whilethe GFP sequence (from the landing pad) was readily detected. More than63x genome coverage was achieved for the TI hosts. To provide additionalassurance of the absence of the antibody A and B in the TI hosts,multiple HCCF samples from TI cell lines expressing antibodies otherthan antibody A and B were evaluated using LC-MS study. The LC-MS datawas analyzed using a similar approach to sequence variant analysis byLC-MS/MS, which has the ability to detect variant peptides at >0.5%level. No antibody A or B peptides were detected in all the samplesharvested from the TI cell lines. Finally, a fluorescence in-situhybridization (FISH) analysis was also performed by Chrombios todetermine the chromosomal location of the GFP landing pad in the genomeof these two TI hosts.

These two well-characterized TI hosts were further assessed for theirRMCE robustness in clinical cell line development with a total of 16different standard antibodies. FIG. 3 shows the productivity of the topclones generated by these TI hosts. Historically the averageproductivity of cell lines generated by random integration was ˜3 g/L.In most cases tested, TI cell lines showed comparable or betterproductivity than RI clones on average.

TI cell lines were expected to have better stability and lower SV thanRI cell lines due to lower antibody gene copy number. For clonestability, monthly fed-batch production was set up monthly for 4 monthsto monitor productivity. Table 3 showed that only 10% of the TI celllines generated by one of the TI hosts had greater than 20% titer dropafter 120 days from PSB. Historically, 60% of the RI cell lines wouldhave similar titer drops. This strongly suggests that the integrationsite identified is highly suitable for stable antibody expression. TIcell lines were also subjected to NAT-based sequence variant analysis.There were significantly lower frequency of SV >5% in the TI cell lineswhen compared with the RI cell lines (4% vs 15%, Table 3).

TABLE 3 Advantages of TI vs RI Characteristics RI TI Copy# Clonedependent Low Clone Stability ~60% of clones ~10% of clones (>20% titerdrop after 120 days from FSB) Sequence Variants (SVs) ~15% of clones ~4%of clones (>5%)

Example 2: Two-Plasmid RMCE Strategy

To address the challenge of achieving high titer expression for standardantibody and expressing multi-chain complex formats, an innovativetwo-plasmid RMCE strategy was developed. The two-plasmid RMCE methodallows 8 or more chains to be targeted to a TI site at the same time.This approach not only increases the flexibility of modulating C and LCchain ratio of antibody to improve productivity, but also enablescomplex molecules with multiple chain as well as targeting transgenes,endogenous genes or RNAi with the antibody to modify cellular pathways.

The two-plasmid RMCE strategy involves using three RRS sites to carryout two independent RMCEs simultaneously (FIG. 4). Therefore, the GFPlanding pad in the TI hosts described above was replaced to include athird RRS site (RRS3) that had no cross activity with either the RRS1 orRRS2 sites. The two expression plasmids to be targeted require the sameflanking RRS sites for efficient targeting, one expression plasmid(front) flanked by RRS1 and RRS3 and the other (back) by RRS3 and RRS2.Since the two-plasmid RMCE efficiency is expected to below, astringentselection scheme is useful to enrich the rare RMCE specific events. Atleast two selection markers are needed in the two-plasmid RMCE. Incertain embodiments three selection markers are employed, which oneselection marker expression cassette split into two parts (see, e.g.,FIG. 4). In certain embodiments involving a split selection markerexpression cassette, the front plasmid would contain the promoterfollowed by a start codon and the RRS3 sequence. The back plasmid wouldhave the RRS3 sequence fused to the N-terminus of the selection markercoding region, minus the ATG start. Additional nucleotides may need tobe inserted between the RRS3 site and the selection marker sequence toensure in frame translation for the fusion protein. Only when the twoplasmids are correctly targeted would the full expression cassette ofthe selection marker be assembled thus rendering cells resistance toselection. FIG. 4 is the schematic diagram showing the two plasmid RMCEstrategy. Of course, single-plasmid RMCE can still be carried out usingRRS1 and RRS2, if needed.

To test the robustness of the two-plasmid RMCE approach, the originalGFP landing pad was replaced by the new GFP landing pad with three RRSsites using a TI host identified previously, Host 4. A more severeviability dip and longer recovery time were observed for the two-plasmidRMCE compared to the single-plasmid RMCE, consistent with lower initialRMCE efficiency for the two-plasmid RMCE. Once the pools recovered,FACS, genomic PCR and gene copy analysis were assessed to confirm bothplasmid cassettes were targeted correctly to the TI site. In total, fivestandard antibodies (Q, R, S, T, and U) were tested for productivityusing both single-plasmid RMCE and two-plasmid RMCE (FIG. 5). Intwo-plasmid RMCEs, more total HC and LC copies were targeted to the TIsite compared to single plasmid RMCE. In all five cases, productivitiesof the two-plasmid TI transfection pools were consistently higher, up to200% increase compared to those of the single plasmid pools. Theincrease in titer in two-plasmid RMCE was mainly attributed to specificproductivity, which saw its level improve by as much as 300% compared tothat of single-plasmid RMCE.

Well-characterized TI hosts, e.g., Host 4, were further assessed in adifferent cell culture platform for their ability to achieve high titersof 5 different antibodies. FIG. 6 shows the productivity of theseantibodies by these TI hosts. Productivity higher of 10 g/L was achievedat Day 14 and higher than 12 g/L at Day 16 for most of the antibodies.

To assess using two-plasmid RMCE to express complex mAb formats, twobispecific molecules that required four different chains (two HCs andtwo LCs) to be expressed in the same cell for bispecific assembly weretested. By enabling two separate expression plasmids to be targetedsimultaneously, plasmid configuration of the different chains can bemanipulated to achieve optimal chain ratios for balanced expression. Inboth cases, cell lines derived from Host 4 were developed having >1.5g/L with >80% bispecific content (FIG. 7) using two-plasmid RMCE.

The day 14 titer of four bispecific molecules that required fourdifferent chains (two HCs and two LCs) to be expressed in the same cellfor bispecific assembly was also evaluated. All the cell lines, whichwere derived from Host 4, were developed having >1.5 g/L titer (FIG. 8)with >80% bispecific content using two-plasmid RMCE.

Example 3: Expression and Growth Analysis of RTI Cell Lines ExpressingmAb-I (Difficult-to-Express) Vs. mAb-II (Average-Expressing) AntibodyMolecules

A difficult-to-express molecule is defined as such when after severalCLD attempts, all the generated cell lines achieve titers that are belowwhat is usually expected from a standard CLD platform process. For onepreviously identified difficult-to-express antibody (mAb-I), forexample, more than 120 cell lines from 4 separate CLD attempts wereevaluated in the fed-batch production cultures. Yet, the highest mAb-Iexpressing cell lines could only achieve 50% of the typical titer whencompared to an average antibody molecule (mAb-II), for which only 24cell lines were screened (FIG. 9A table). Unfortunately, tracing theunderlying cause(s) that might make a molecule difficult-to-express in arandom integration system is very difficult, mainly due to thedifferences in transgene copy numbers and gene integration sites betweendifferent cell lines.

To identify the underlying factor(s) that made mAb-Idifficult-to-express, CLD was initiated for both mAb-I and mAb-IImolecules using a RTI system. Both HC and LC constructs were cloned intoan expression vector under the inducible CMV-TO promoter control andthese vectors were transfected into a TI host in order to increase thepossibility of isolating cell lines with similar levels of transgenetranscription. The overall schematic of the RTI CLD approach for mAb-Iand mAb-II is depicted in FIG. 9B. Transfected cell lines were subjectedto selection using two separate conditions, under which cell lines wereeither 1) derived in the presence of Dox (Induced), or 2) in the absenceDox (Not-Induced), throughout the CLD process. Cell lines derived in theabsence of Dox were only temporarily induced to be ranked based on seedtrain titer or during production assays where indicated. This enabledthe evaluation of the role of antibody expression (expression pressure)during the selection process (FIG. 9B).

Production assays were performed using two representative mAb-I andmAb-II cell lines, derived from Host 7, from both “Induced” and“Not-Induced” seed-train arms in the presence or absence of doxycycline.The latter was used to evaluate promoter leakiness and basal antibodyproduction levels in the absence of induction. A rather tight regulationof antibody expression in the absence of doxycycline was observed,suggesting that the regulated expression system functioned effectivelyin these cell lines (FIGS. 10A and 10C). Growth (FIG. 10B) profiles ofall cell lines in the presence or absence of doxycycline werecomparable, although in the absence of doxycycline growth profilesappeared slightly better. Cell lines that were derived under “Induced”or “Not-Induced” seed-train conditions displayed relatively comparabletiters and specific productivities during production (FIGS. 10A and 10C,compare cell lines 1&2 to 3&4 for mAb-I or mAb-II), indicating thatconstitutive antibody expression during selection did not play asignificant role in isolation of high titer cell lines (FIG. 10). Inaddition, similar to the RI CLD attempts, titer and specificproductivity of mAb-I RTI cell lines were still 50% lower than mAb-IIRTI cell lines (FIGS. 10A and 10C). This observation ruled out chronictoxicity as a culprit for low expression of mAb-I, since both “Induced”and “Not-Induced” RTI cultures of mAb-I similarly displayed lower titerscompared to the mAb-II control cell lines.

Example 4: Lower Antibody Expression in mAb-I Cell Lines was not Due toLower Transcription

To analyze lower antibody expression observed in mAb-I expressing celllines, qRT-PCR experiments were performed to measure mRNA levels ofantibody HC and LC in the RTI cell lines expressing mAb-I or mAb-II,under doxycycline induced and uninduced conditions. In the absence ofDox, mAb-I or mAb-II cell lines expressed little or no HC or LC mRNA,indicative of a relatively tight transcriptional regulation by theinducible promoter (FIG. 11). In the presence of Dox, antibody HC and LCtranscription levels for mAb-I expressing cell lines were comparable toor higher than those of mAb-II expressing cell lines (FIGS. 11A and11B). These results confirmed that a reduced level of transgenetranscription was not the reason for low antibody expression in mAb-Iexpressing cell lines. Additionally, absence or presence of doxycyclinein the seed-train cultures during CLD process had no impact on transgenetranscription rates, since cell lines isolated under “Induced” or“Not-Induced” conditions had comparable HC or LC mRNA levels (FIGS. 11Aand 11B).

Example 5: Induction of mAb-I Expression Resulted in ReversibleAccumulation of Intracellular BiP and Delayed Antibody HC Degradation

To identify the underlying factors affecting mAb-I expression, Doxinduction along with cycloheximide (CHX) treatment and removal was usedto investigate antibody secretion, folding and degradation. For theseexperiments, “Induced” cultures were treated with CHX to stop proteinsynthesis and after 5 hours, cells were washed and resuspended intomedia containing only Dox (no CHX), to resume protein synthesis (FIG.12A). HC or LC were not detected in the supernatants of these culturesfor approximately 60 min post CHX removal and a gradual increase inantibody secretion was only detected after 120 min and at similar rates(FIG. 12B, upper panels). This suggested that secretion of properlyfolded and assembled antibody molecules were comparable between mAb-Iand mAb-II cell lines post CHX removal. The intracellular levels ofantibody LC were also fairly comparable between mAb-I and mAb-IIexpressing cell lines (FIG. 12B, LC panels). However, significantlyhigher levels of intracellular antibody HC in mAb-I expressing celllines were detected compared to mAb-II expressing lines, even after 5hours of CHX treatment (FIG. 12B, HC panels). This implicated a delay orproblem in degradation and clearance of unfolded or mis-folded antibodyHC in mAb-I expressing cell lines. Interestingly, very high levels ofintracellular BiP in the mAb-I were detected relative to the mAb-II celllines before and after CHX treatment (FIG. 12B, lower panels, BiP). BiPas a chaperone has been implicated in the unfolded protein response(UPR) as well as antibody HC or LC folding, and its presence in the seedtrain culture could be an indicator of ER associated cellular stress.

To further investigate the correlation between mAb-I expression andintracellular BiP accumulation, “Induced” seed train cultures weretreated with or without CHX overnight in media containing doxycyclineand evaluated the intracellular HC, LC and BiP levels in these samples.In the absence of CHX treatment, the basal levels of intracellularantibody HC molecules were higher in mAb-I relative to mAb-II celllines, while levels of intracellular LC were comparable between allsample sets. BiP levels on the other hand were considerably higher inmAb-I relative to mAb-II expressing cell lines (FIG. 12C, -CHX samples).Overnight treatment of these cultures with CHX resulted in a reductionin levels of HC and LC in all cell lines, along with a concurrentreduction in BiP levels specifically in mAb-I expressing cell lines(FIG. 12C, +CHX samples). This was in agreement with previous findings(FIG. 12B). Since cycloheximide inhibits synthesis of all eukaryoticproteins indiscriminately, the higher BiP levels in mAb-I cell linescould not be directly linked to accumulation of antibody HC or LCsubunits.

However, the regulated expression system offered the ability to directlytest the link between intracellular BiP and antibody HC or LC levels. Tothat end doxycycline was removed from the media for 6 days, tospecifically turn off antibody expression without affecting expressionof other endogenous proteins, and monitored intracellular HC, LC, andBiP levels. A direct and specific link between antibody (HC and LC)expression and accumulation of intracellular BiP for mAb-I cell lineswas observed, while expression of mAb-II showed no effects onintracellular BiP levels (FIG. 12D).

Example 6: Although mAb-I LC Expression Triggered Intracellular BiPAccumulation, mAb-I HC Also Distinctly Contributed to Low Expression ofthis Antibody

To determine whether mAb-I LC played a role in accumulation ofintracellular BiP, a construct was generated that expressed mAb-I HCunder a regulated promoter while LC was expressed constitutively. Thisconstruct along with constructs expressing regulated HC/LC of mAb-I ormAb-II (controls) were transfected into the CHO cells and two separatepools from each transfection were derived. Pool titers were evaluated inproduction culture (FIG. 13A) while seed train cultures were evaluatedby Western blot analysis (FIG. 13B) in the presence and absence ofdoxycycline. During production assay, each pool was induced with Dox orremained uninduced. Uninduced pools expressed very little antibodyduring production, indicative of a tight expression regulation (FIG.13A, -Dox). Analysis of Dox induced cultures during production revealedthat the regulated-HC/constitutive-LC mAb-I expressing pools derivedfrom “Not-induced” seed train cultures had considerably lower titersrelative to the regulated HC/LC mAb-I pools andregulated-HC/constitutive-LC mAb-I pools that were “Induced” during seedtrain (FIG. 13A). Western blot analysis of “Not Induced” cultures showedthat under all conditions antibody HC was expressed only when Dox wasadded to the media. In addition, constitutive expression of mAb-I LCalone, without the mAb-I HC, triggered accumulation of intracellular BiP(FIG. 13B). These results implied a direct connection between expressionof mAb-I LC subunit and accumulation of the intracellular BiP. Whenconstitutively expressed in the absence of HC, the LC molecules of mAb-Ilikely form unfolded aggregates within the ER that interact with BiPchaperone, triggering an increase in intracellular levels of BiP inthese cells. Expression of mAb-I HC during production phase could havefurther burdened these cells, increasing the demand for intracellularBiP to accommodate folding of both HC and LC molecules, resulting ininefficient antibody assembly, folding and secretion efficiency (FIG.13A).

While these findings pointed to a connection between mAb-I LC expressionand cellular ER stress, as indicated by accumulation of intracellularBiP, a direct connection between expression of mAb-I LC and low titersof mAb-I cell lines needed to be established. To that end, antibodymolecules were generated where mAb-I and mAb-II heavy and light chainswere swapped to generate HC_(I)LC_(I), HC_(I)LC_(II), HC_(I)LC_(II), andHC_(II)LC_(I) antibody combinations. HC_(I)LC_(II) construct expressedan antibody with mAb-I HC and mAb-II LC, while HC_(II)LC_(I) constructexpressed an antibody with mAb-II HC and mAb-I LC. Antibody HC and LCexpression for all these constructs were under the control of CMV-TOpromoter in the RTI vector system (FIG. 13C). These constructs were thentransfected into our CHO TI host cells and two separate RTI pools, foreach construct, were generated and expression of mAb-I, or mAb-II, orthe hybrid versions of these antibodies were tested. As previouslynoticed (FIG. 13B), a clear and direct correlation between mAb-I LCexpression and accumulation of intracellular BiP was observed (FIG. 13D,LC and BiP blots). Interestingly, intracellular BiP was also accumulatedin a less intense but yet noticeable manner in pools expressingHC_(I)LC_(II) hybrid antibody compared to HC_(II)LC_(I), hinting towardssome levels of ER stress in these pools, likely due to expression ofHC_(I)(FIG. 13D, compare long exposure BiP levels in HC_(II)LC_(II) andHC_(I)LC_(I) samples). Turning off antibody expression via Dox removalresulted in reduction of intracellular BiP levels in all RTI poolsexcept for mAb-II (HC_(B)LC_(B)) pool, where the initial levels of bothintracellular HC and BiP were considerably lower even prior to Doxremoval (FIG. 13D, compare HC and BiP levels at Day 0 in HC_(II)LC_(II)pools to other pools).

To evaluate the role of mAb-I HC and LC on overall antibody expression,the HC_(I)LC_(I), HC_(II)LC_(II), HC_(I)LC_(II), and HC_(II)LC_(I) RTIpools were evaluated in production assays.

In the absence of doxycycline, all the RTI pools expressed littleantibody, exhibiting a tight regulation of antibody expression (FIG.14A, -Dox). Growth rates for all the RTI pools were similar, with theinduced cultures having slightly lower growth rates relative to theuninduced cultures (FIG. 14B). As previously observed, the mAb-I andmAb-II RTI pools had the lowest and highest titers and specificproductivities, respectively (FIGS. 14A and 14C). Interestingly, bothHC_(II)LC_(I) and HC_(I)LC_(II) RTI pools displayed titers and specificproductivities that were higher than HC_(I)LC_(I) but lower thanHC_(II)LC_(II) RTI pools (FIGS. 14A and 14C). These results indicatedthat both mAb-I HC and LC subunits individually contributed to the lowerexpression of this antibody relative to the mAb-II. Contrary to ourexpectations, hybrid RTI pools that expressed LC_(I) subunit hadslightly higher titers and specific productivities compared to the poolsexpressing HC subunit (FIGS. 14A and 14C). This indicated that mAb-I HCplayed a slightly more significant role in the lower expression of thisantibody relative to the LC subunit. It was the use of antibody chainswap approach in combination with the RTI system that revealed thenegative effects associated with the expression of mAb-I HC subunit,even when a clear indicator of biological stress (such as intracellularBiP accumulation) could not be identified.

Example 7: Amino Acid Sequence Differences Between mAb-I and mAb-II HCand LC Subunits

To assess whether certain amino acids residues might contribute to theproblematic expression of mAb-I HC and LC, the amino acid sequences ofmAb-I and mAb-II LC (FIG. 15A) and HC (FIG. 15B) subunits were aligned.The amino acid composition for all CDR regions of mAb-I and mAb-II HCand LC molecules were different from each other, as they were developedto target different antigens. In the LC subunit, there are no amino acidsequence differences between mAb-I and mAb-II molecules for the rest ofthe variable and all of the constant regions. The CDR1 segment of LC_(I)is 6 amino acids longer than LC_(II), while CDR3 segment of LC_(II) isone amino acid longer than LC_(I) (FIG. 15A). As for the antibody HCsubunit, excluding the CDR segments, the rest of the variable andconstant regions of HC_(I) and HC_(II) are identical with the exceptionof 4 amino acids residues (FIG. 15B). Two of these amino acid changeswere fairly conservative (T->S and L->A) and hence unlikely tocontribute to the low expression of HC_(I). The N->G change in HC_(I)prevents antibody HC glycosylation. Hence, the effects of N->G and Y->Vamino acid differences between HC_(II) and HC_(I) on expression ofHC_(I)were investigated. Two point mutations were generated, changingthe G and V amino acid residues of HC_(I) to N and Y, respectively (HCAdouble mutant or HC_(I)dm). The HC_(I)dm was used along with LC_(I) andLC_(II) to generate RTI pools and antibody expression in these poolswere evaluated in production cultures (FIG. 16A). The findings showedthat co-expression of HC_(I)dm along with LC_(I) increased antibodyexpression to the levels observed for HC_(II)LC_(II), suggesting thatthese mutations can restore the low expression levels of HCA antibody(FIG. 16A). Interestingly, when co-expressed with LC_(II), HC_(I)dmachieved titers even higher than that of HC_(II)LC_(II), suggesting thatHC_(I)dm is superior in expression and folding compared to HC_(II) (FIG.16A). As previously observed, HC_(I)LC_(I) had the lowest titer whileHC_(I)LC_(II) displayed an intermediate antibody expression profile(FIG. 16A). Western blot analysis revealed that expression of LC_(I) wasstill associated with increased intracellular BiP accumulation, evenwhen co-expressed with HC_(I)dm (FIG. 16B). FIG. 16C depicts adiagnostic roadmap of how RTI system can be used to dissect and identifythe source of problem in cell lines that express hard to expressmolecules.

Example 8: Generation of TI Host Cell Containing Two Distinct andIndependent Landing Pads

Host 8 is a targeted integration (TI) host containing two distinct andindependent landing pads, which can be targeted for expression ofantibody genes individually, or simultaneously. The parental cell linefor Host 8 is Host 7 (see Table 2) which contains a single landing pad.A second landing pad was inserted into Host 8 at the genomic locationwhere the landing pad is located in the Host 4 TI host (see Table 2).This insertion was performed by CRISPR/Cas9-based knock-in of a newlanding pad into the Host 4 TI site location within the Host 7 TI host.

To accomplish this, the genomic region of Host 4 TI site wascharacterized within the TI Host 4 and the TI Host 7. Based on thisinformation, a guide RNA was constructed to target this specific genomicregion to facilitate CRISPR/Cas9-based knock-in. Separately, a donorplasmid containing a newly created landing pad was generated to beknocked-in to this site and constructed together with homology armscorresponding to the genomic region upstream and downstream of thetargeted knock-in site to facilitate knock-in, and additional genes toallow for screening of knock-in clones. Co-transfection of a plasmidcontaining genes for the gRNA and Cas9, and the donor plasmid wereperformed to facilitate the CRISPR/Cas9-based knock-in of the newlanding pad.

Following selection and screening of clones that underwentCRISPR/Cas9-based knock-in, Host 8 was ultimately identified andunderwent further evaluation. The existing Host 7 landing pad wasdetermined to be intact, and the newly knocked-in landing pad at theHost 4 site location was also confirmed to be present, intact, and inthe correct location.

The feasibility of recombinase-mediated cassette exchange (RMCE) at bothsites was then evaluated for each site individually, and thensimultaneously. Clones were generated by RMCE at the Host 7 landing padonly, the Host 4 landing pad only, and both sites simultaneously, andthen evaluated in a 14-day fed-batch shake flask evaluation. Titer andspecific productivity of the clones expressing antibody at both sitessimultaneously was higher compared to expression at each siteindividually. Further evaluation was performed in a different cellculture process, cell culture platform B, to show additional titerincrease for all clones in comparison to the cell culture platform A(FIG. 17). The average 14-day titer for the Host 4 site was 3.0 g/L, forthe Host 7 site was 1.9 g/L and for both sites simultaneously was 3.9g/L at the platform A cell culture.

Ultimately, TI Host 8 was created from TI Host 7 by addition of a secondlanding pad located at the genomic location of the landing pad of the TIHost 4. Further investigation confirms knock-in of the landing pad andfeasibility of RMCE at both, existing and new, landing pads. Further,evaluation of clones generated from Host 8 at each site individually andboth sites simultaneously show productivity of antibody expression. Theexpression of antibody genes at both sites simultaneously, versus eachindividual site, have an additive productivity effect which is seen inboth the cell culture platform A and in the cell culture platform B(FIG. 17).

Methods

Antibody Plasmid DNA Construct Configurations: To construct one-plasmidantibody constructs, antibody HC and LC fragments were cloned into avector backbone containing the L3 and 2 L sequences as well as apuromycin N-acetyl-transferase (pac) selectable marker. To constructtwo-plasmid antibody constructs, antibody HC and LC fragments werecloned into a front vector backbone containing L3 and LoxFAS sequences,and a back vector containing LoxFAS and 2 L sequences and apacselectable marker. The Cre recombinase plasmid pOG231 (Wong E T et al.,Nucleic Acids Res 2005, 33, (17), 5 e147; O'Gorman S et al., Proc NatlAcad Sci USA 1997, 94, (26), 14602-7, the contents of each areincorporated herein by reference) was used for all RMCE processes.

Cell Culture: CHO cells were cultured in a proprietary DMEM/F12-basedmedium in 125 mL shake flask vessels shaking at 150 rpm, 37° C. and 5%CO₂. Cells were passaged at a seeding density of 3×10⁵ cells/mL every3-4 days.

RMCE Stable Cell Line Development: Expression plasmids were transfectedinto TI hosts by MaxCyte STX® electroporation (MaxCyte, Gaithersburg,Md.). RMCE transfection pools were then selected with puromycin and1-(2′-deoxy-2′-fluoro-1-beta-D-arabinofuranosyl-5-iodo)uracil (FIAU)(Moravek). After pool selection, single cell cloning (SCC) was performedto generate TI cell lines for further evaluation.

Fed-Batch Production Assay: Fed-batch production cultures were performedin shake flasks or ambr15 vessels (Sartorius Stedim) with proprietarychemically defined production medium. Cells were seeded at 1×10⁶cells/ml on day 0, with a temperature shift from 37° C. to 35° C. on day3. Cultures received proprietary feed medium on days 3, 7, and 10.Viable cell count (VCC) and percent viability of cells in culture wasmeasured on days 0, 3, 7, 10, and 14 using a Vi-Cell™ XR instrument(Beckman Coulter). Glucose and lactate concentrations were measured ondays 7, 10 and 14 using a Bioprofile 400 Analyzer (Nova Biomedical). Day14 titers were determined using protein A affinity chromatography withUV detection.

Gene copy number determination by droplet PCR: Droplet PCR assays wereperformed using ddPCR™ Supermix kit (Bio-Rad). Each ddPCR reactioncontaining ddPCR Master mix, 900 nM forward primer, 900 nM reverseprimer, 250 uM probe, 3 unit HaeIII restriction enzyme and sample DNA.After incubation at room temperature for 10 min, the droplets weregenerated with Automatic Droplet Generator (Bio-Rad). The PCR thermalcycling conditions were 10 min at 95° C., followed by 40 cycles of 94°C. for 30 sec and 60° C. for 1 min, then 98° C. for 10 min to deactivatethe enzyme. After PCR reactions, the droplets were read on the QX200™Droplet reader (Bio-Rad). The data were collected and analyzed usingQuantasoftware. The HC and LC gene copy numbers were normalized based onthe defined copy number of reference genes Bax, Albumin, Hprt andb-microglobulin. The primers used in this study were designed usingPrimer Express v3.0 (Life Technologies). The sequences of the primersare listed in Table 4.

TABLE 4 Primer sequences for droplet PCR. Primers SequenceHeavy Chain - Forward Primer TCA AGG ACT ACT TCC CCG AAC CHeavy Chain - Reverse Primer TAG AGT CCT GAG GAC TGT AGG ACA GCHeavy Chain - Probe VIC-ACG GTG TCG TGG AAC TCA GGC GC-TAMRALight Chain - Forward Primer GCT GCA CCA TCT GTC TTC ATC TLight Chain - Reverse Primer GCA CAC AAC AGA AGC AGT TCC ALight Chain - Probe VIC-CCC GCC ATC TGA TGA GCA GTT GAA-TAMRABax - Forward Primer ACA CTG GAC TTC CTC CGA GA Bax - Reverse PrimerGCA TTA GGA AGT TTG AGA ACC A Bax - Probe FAM-CCC AGC CAC CCT GGT CTT GG-TAMRA Albumin - Forward Primer TTC GTG ACA GCT ATG GTG AAC TGAlbumin - Reverse Primer GGT CAT CCT TGT GTT TCA GGA AA Albumin - ProbeFAM-CTG TGC AAA ACA AGA ACC CGA AAG AAA CC-TAMRA Hprt - Forward PrimerAAA GGA CAT AAT TGA CAC TGG TAA A Hprt - Reverse PrimerCAA TTG CTT ATT GCT CCC AA Hprt - Probe FAM-CTG CTT TCC CTG GTC AAG CGG-TAMRA β-microglobulin - Forward Primer CTT CCT GAA CTG CTA TGT GTC TCA Aβ-microglobulin - Reverse Primer CCA TCT TCT TTC CAT TCT TCA ACAβ-microglobulin - Probe VIC-TTC ATC CCC CCC AAA TTG AAA TCG A-BHQ1

Quantitative Real Time PCR (qRT-PCR or Tagman) Analysis: RNA samplesfrom seed train cultures were purified using Qiagen RNeasy plus mini kitaccording to the manufacturer's instructions. TaqMan RT-PCR assays wereperformed using TaqMan One® Step RT-PCR Master mix kit (LifeTechnologies). Each RT-TaqMan PCR reaction contained RT-PCR Master mix,reverse transcriptase, 300 nM forward primer, 300 nM reverse primer, 100nM probe, and 10 ng purified RNA sample. The thermal cycling conditionswere 30 min at 48° C. and 10 min at 95° C., followed by 40 cycles of 95°C. for 15 sec and 60° C. for 1 min. All reactions were processed on the7900 HT Fast Real Time PCR System (Life Technologies). Data wereanalyzed using SDS v2.4 software (Life Technologies) after TaqMan RT-PCRamplification. The relative expression levels of HC and LC weredetermined based on the delta Ct analysis method. The expression levelof a house keeping gene, cyclophilin, was used as reference gene tonormalize different RNA samples in each reaction. The primers used inthis study were designed using primer express v3.0 (Life Technologies).The sequences of primers are listed in Table 5.

TABLE 5 Primer sequences for Quantitative Real Time PCR Primers SequenceHeavy Chain - Forward Primer TCA AGG ACT ACT TCC CCG AAC CHeavy Chain - Reverse Primer TAG AGT CCT GAG GAC TGT AGG ACA GCHeavy Chain - Probe FAM-ACG GTG TCG TGG AAC TCA GGC GC-TAMRALight Chain - Forward Primer TGA CGC TGA GCA AAG CAG ACLight Chain - Reverse Primer CAG GCC CTG ATG GGT GAC Light Chain - ProbeFAM-ACG AGA AAC ACA AAG TCT ACG CCT GCG A-TAMRAHamster Cyclophilin - Forward Primer GCG TTT CGG GTC CAG GAHamster Cyclophilin - Reverse Primer GAG TTG CCC ACA GTC GGA AHamster Cyclophilin - Probe FAM-TGG CAA AAC CAG CAA GAA GAT CAC CA-TAMRA

Western Blot analysis: Cell pellets from seed train cultures were washedwith PBS and resuspended in NP-40 lysis buffer with the mini cOmplete™cocktail of protease inhibitors (Roche). Cell lysates were centrifugedat 13000 RPM for 10 minutes and the supernatants were collected. Proteinconcentration for each lysate was determined using the Pierce BCAProtein Assay Kit (Thermo Scientific). Equal protein concentrations ofeach lysate were mixed with SDS-PAGE buffer and NuPage Sample ReducingAgent (Invitrogen) before being heat-denatured for 5 minutes at 95° C.The lysates were then loaded onto NuPage precast 4-12% Bis-Tris gels(Invitrogen) and electrophoresed. Protein was transferred ontonitrocellulose membranes using the iBlot system (Invitrogen). Themembranes were blocked for 1 hour at room temperature using 5% nonfatmilk solution in tris-buffered saline containing 0.1% Tween 20 (TBS-T).After blocking, membranes were incubated for 1 hour at room temperatureusing specific primary antibodies and then washed 3 times for 10 minuteswith TBS-T. Secondary horseradish peroxidase conjugated antibody wasadded for 1 hour at room temperature, followed by 3 15-minute washeswith TBS-T. Membranes were developed using either the Amersham ECLdetection reagent or Amersham ECL Prime detection reagent (GE LifeSciences). Image Lab 5.1 (Biorad) was used for band signalquantification. The following primary antibodies were used: 1:3000 goatanti-human IgG-HRP (MP Biomedicals) and 1:2500 mouse anti-P actin (SigmaAlrdich). The following secondary antibody was used: 1:10000 sheepanti-mouse (GE Healthcare UK).

Example 9: Addition of an Extra Light Chain to the One Heavy, One LightChain Plasmid DNA Configuration Improves Antibody Titer and SpecificProductivity

To examine the effect of antibody copy number on productivity in the TIhost, RMCE pools were generated by transfecting a single plasmidcontaining either one heavy and one light chain (HL configuration) orone heavy and two light chains (HLL configuration). After selection,recovery, and verification of RMCE by flow cytometry, the pools'productivity was evaluated in a 14-day fed batch production assay. Forthree separate antibodies (mAb-Q, mAb-S, and mAb-T) an approximately2-fold increase in titer for the HLL pools compared to the HL pools wasobserved, driven largely by a 1.5-2.5-fold increase in specificproductivity (FIGS. 18A and 18B). Additional single cell clones weregenerated from the transfected pools by using limiting dilution andconfirmed that titer and specific productivity are also higher in HLLconfiguration derived clones than those from the HL configuration (FIG.18C, 18D).

Example 10: Transfection with Up to Seven Antibody Chains in RMCE PoolsIncreases Antibody Specific Productivity and Titer

The effect of increasing the antibody chain number on expression of theantibody was evaluated. The HLL single plasmid configuration wascompared to the HLL-HL or HLL-HLL dual plasmid configurations for fiveantibodies.

The HLL-HL (five-chain), HLL-HLL (six-chain) and HLL-HLHL (seven-chain)configurations for mAb Y were compared. Titer and Qp of RMCE pools inproduction generally increased from the HLL-HL to HLL-HLL and HLL-HLHLconfigurations (FIG. 19A, 19B), while growth (represented as theintegral of viable cell count, IVCC) among the different pools wasrelatively equivalent (FIG. 19C). Looking at seed train antibody mRNAexpression in these pools, an increase in light chain mRNA going fromHLL-HL to HLL-HLL and HLL-HLHL, as well as increased heavy chain mRNA inthe HLL-HLHL pool compared to HLL-HL and HLL-HLL pools was observed(FIG. 19D). The intracellular protein levels of heavy chain and lightchain from these RMCE pools were also measured using western blot.Intracellular light chain protein levels closely matched what wasobserved for light chain mRNA, with increases from the five to six andseven chain configurations (FIGS. 19E, 19F).

Example 11: Single Cell Clones with the Seven Chain HLL-HLHLConfiguration have High Titer and Specific Productivity in Two TestedAntibodies

To evaluate whether the high titer and productivity of the seven chainconfiguration HLL-HLHL is maintained after single cell cloning, singlecell monoclones from the HLL-HL, HLL-HLL, and HLL-HLHL RMCE pools of mAbY were generated. These monoclones were then evaluated in a 14 dayfed-batch production assay. Looking at the top 6 clones from eachconfiguration, it was found that titer and Qp increased slightly fromthe HLL-HL configuration to HLL-HLL and HLL-HLHL (FIG. 20A, 20B), asgrowth remained similar (FIG. 20C).

To confirm that the HLL-HLHL configuration results in high monoclonetiter, a different mAb, mAb III was used to generate single cell clones,derived from Host 4, from HLL-HL (five-chain) and HLL-HLHL (seven-chain)RMCE pools. After evaluation in a 14 day fed batch production assay, aclear titer advantage of the HLL-HLHL clones over the HLL-HL clones wasobserved (FIG. 20D), greater than that seen with mAb U. The titerincrease appeared to be driven primarily by the higher Qp of theHLL-HLHL clones (FIG. 20E), with little average difference in IVCCbetween the two configurations (FIG. 20F).

Example 12: Antibody Chain Position in a Transfected Plasmid Affects itsProductivity

The effect of the position or arrangement of heavy chain and light chainin the transfected plasmids on antibody expression was also evaluated.To do this, RMCE pools were generated with plasmids containing severaldifferent configurations of one heavy chain and one light chain of mAbZ. Since the TI hosts of the present disclosure support integration oftwo different plasmids at the same integration site, the position effectwhen both heavy chain and light chain are expressed from the sameplasmid as well as when they are separated on either front or backdifferent plasmids was examined (FIG. 21A).

After generating the RMCE pools, the mRNA expression level of antibody Zheavy chain and light chain in seed train cultures were measured. ThemRNA expression of heavy chain decreased when it was preceded by lightchain, with the LH configuration in both front and back plasmids havinglower heavy chain mRNA expression than the HL configuration. However,the effect of position on light chain was dependent on which plasmid itwas expressed from: in the front plasmid, light chain mRNA expressionincreased in the LH configuration vs. HL but the opposite was true inthe back plasmid. It was also observed that while a light chain cassetteon its own in the front plasmid expressed a high level of light chainmRNA, a single light chain cassette in the back plasmid had the lowestlight chain mRNA levels of any configuration (FIG. 21). In general,lower mRNA expression levels for both heavy and light chain in the backplasmid compared to the front plasmid were observed.

A fed-batch production assay of these different RMCE pools produced arange of titers and specific productivities similar to the trend inantibody mRNA expression (FIG. 21C, 21D). The LH-no mAb configurationhad higher Qp and titer than HL-no mAb, while the pools with the lowestlevel of LC mRNA expression—H-L and no mAb-LH—also had the lowest Qp andtiter.

To confirm that the observed mRNA and antibody productivity differencesare due to antibody chain position and not variation in absolute copynumber, digital droplet PCR was used to measure the pools' heavy andlight chain copy number. As expected, all pools had approximately oneheavy and one light chain DNA copy (FIG. 21E).

Example 13: Effect of HC to LC Ratio on the % High-Molecular-WeightSpecies on mAbs

The effect of the HC:LC ratio of antibodies on productivity and onHigh-molecular-weight species (HMWS), such as aggregates, was evaluated.Furthermore, the effect on the TI site was evaluated by using cells withdifferent TI sites. To do this, RMCE pools were generated bytransfecting a single plasmid containing either one heavy and one lightchain (HL configuration) or one heavy and two light chains (HLLconfiguration) in TI Host 4 or Host 7 locations. The pools' productivitywas evaluated in a 14-day fed batch production assay (FIGS. 22A and22B). The % HMWS was evaluated (FIGS. 22B and 22D) showing that theeffect of HC:LC ratio is independent of the integration site.

The effect of increasing the LC copy of and antibody on the productivityand on High-molecular-weight species (HMWS) was evaluated, in connectionwith mAb IV. To do this, RMCE pools were generated by transfecting asingle plasmid containing either one heavy and one light chain (HLconfiguration), one heavy and two light chains (HLL configuration), twoheavy and three light chains (HLL-HL configuration), two heavy and fourlight chains (HLL-HLL configuration), two heavy and five light chains(HLL-HLLL configuration), three heavy and 6 light chains (HLLL-HLLHLconfiguration), or three heavy and 7 light chains (HLLL-HLLHLLconfiguration) in Host 4 site location or in Host 4 and 7 site location(FIGS. 23 and 24).

Through a number of titer and production evaluation assays, a number ofclones were identified and evaluated in an ambr15 production evaluationwith a specific production process. From this evaluation, the top 10clones, identified by cell culture performance, titer, and productquality attributes (including percent aggregation), were chosen to bebanked for further evaluation. Ultimately, 2-site integration cell lineswere generated expressing mAb-VI with high titer, lower percentaggregation, and comparable production quality to previously generatedmAb-VI cell lines. FIG. 25 shows the top 2 clones with titers of 8.4 and8.7 g/L with 7.4% and 5.6% aggregates, respectively.

Example 14: Regulated Targeted Integration: Clone Evaluation

In this experiment the expression of antibody Z, in a regulatedderivative of Host 4, was evaluated after the regulated targetedintegration of a sequence encoding the antibody was performed. Thesystem employed for the regulation of the expression of antibody Z iscomposed of 3 key components: 1) Regulated promoters that include tandemtet-operon sequences prior to a signal sequence, 2) Promoter andTet-repressor protein coding sequences, and 3) an inducer, such asdoxycycline, to allow for expression of antibody Z.

Upon confirmation that targeted integration of the sequence encodingantibody Z, clone evaluation was performed in a shake flask process A10% feed (of the total volume) on days 3, 7 and 10 was performed. Oneproduction culture assay was carried out to evaluate the clones. Allclones were inoculated at a seeding density of 3×10⁶ cells/mL on day 0.On days 3, 7, and 10, 1 μg/mL doxycycline was added to all 36 culturesto induce expression of antibody Z.

Final titers and specific productivity (Qp) are shown in FIGS. 26 and 27respectively. Day 14 Harvest Cell Culture Fluid (HCCF) was submitted totiter analysis. Titers ranged from 1 to 3.68 g/L (excluded clone 7).Clone selection for banking was first ranked by titer and finalselection was based on cell culture performance, including Qp and otherparameters. The selected clones for banking produced titers ranging from2.69 to 3.68 g/L.

In addition to the various embodiments depicted and claimed, thedisclosed subject matter is also directed to other embodiments havingother combinations of the features disclosed and claimed herein. Assuch, the particular features presented herein can be combined with eachother in other manners within the scope of the disclosed subject mattersuch that the disclosed subject matter includes any suitable combinationof the features disclosed herein. The foregoing description of specificembodiments of the disclosed subject matter has been presented forpurposes of illustration and description. It is not intended to beexhaustive or to limit the disclosed subject matter to those embodimentsdisclosed.

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions and methodsof the disclosed subject matter without departing from the spirit orscope of the disclosed subject matter. Thus, it is intended that thedisclosed subject matter include modifications and variations that arewithin the scope of the appended claims and their equivalents.

Various publications, patents and patent applications are cited herein,the contents of which are hereby incorporated by reference in theirentireties.

1-77. (canceled)
 78. A targeted integration (TI) host cell comprising an exogenous nucleotide sequence integrated at an integration site within a specific locus of the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from: contig NW_006874047.1; contig NW_006884592.1, contig NW_006881296.1, contig NW_003616412.1, contig NW_003615063.1, contig NW_006882936.1, and contig NW_003615411.1.
 79. The TI host cell of claim 78, wherein the nucleotide sequence immediately 5′ of the integrated exogenous nucleotide sequence is selected from the group consisting of (a) sequences at least about 90% homologous to nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 of NW_003615411.1; or (b) sequences at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, at least 3,000 base pairs from nucleotide 45269 of NW_006874047.1, nucleotide 207911 of NW_006884592.1, nucleotide 491909 of NW_006881296.1, nucleotide 79768 of NW_003616412.1, nucleotide 315265 of NW_003615063.1, nucleotide 2662054 of NW_006882936.1, and nucleotide 97705 of NW_003615411.1.
 80. The TI host cell of claim 78, wherein the nucleotide sequence immediately 3′ of the integrated exogenous nucleotide sequence is selected from the group consisting of (a) sequences at least about 90% homologous to nucleotides 45270-45490 of NW_006874047.1, nucleotides 207912-792374 of NW_006884592.1, nucleotides 491910-667813 of NW_006881296.1, nucleotides 79769-100059 of NW_003616412.1, nucleotides 315266-362442 of NW_003615063.1, nucleotides 2662055-2701768 of NW_006882936.1, and nucleotides 97706-105117 of NW_003615411.1; or (b) sequences at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, at least 3,000 base pairs from nucleotide 45270 of NW_006874047.1, nucleotide 207912 of NW_006884592.1, nucleotide 491910 of NW_006881296.1, nucleotide 79769 of NW_003616412.1, nucleotide 315266 of NW_003615063.1, nucleotide 2662055 of NW_006882936.1, and nucleotide 97706 of NW_003615411.1.
 81. The TI host cell of claim 78, wherein the integrated exogenous nucleotide sequence is operably linked to a nucleotide sequence selected from the group consisting of sequences at least about 90% homologous to a: contig NW_006874047.1; contig NW_006884592.1, contig NW_006881296.1, contig NW_003616412.1, contig NW_003615063.1, contig NW_006882936.1, and contig NW_003615411.1.
 82. A targeted integration (TI) host cell comprising an exogenous nucleotide sequence integrated at an integration site within or operably linked to an endogenous gene selected from the group consisting of LOC107977062, LOC100768845, ITPR2, ERE67000.1, UBAP2, MTMR2, XP_003512331.2, and sequences at least about 90% homologous thereto.
 83. The TI host cell of claim 78, wherein the TI host cell is a mammalian host cell.
 84. The TI host cell of claim 83, wherein the TI host cell is a hamster host cell, a human host cell, a rat host cell, or a mouse host cell.
 85. The TI host cell of claim 78, wherein the exogenous nucleotide sequence comprises one or more recombination recognition sequences (RRSs), wherein the RRS can be recognized by a recombinase.
 86. The TI host cell of claim 85, wherein the exogenous nucleotide sequence comprises at least two RRSs.
 87. The TI host cell of claim 85, wherein the recombinase is a Cre recombinase, an FLP recombinase, a Bxb1 integrase or a (pC31 integrase.
 88. The TI host cell of claim 86, wherein the exogenous nucleotide sequence comprises a first and a second RRS, and at least one selection marker located between the first and the second RRS.
 89. The TI host cell of claim 88, further comprising a second selection marker, wherein the first and the second selection markers are different.
 90. The TI host cell of claim 89, further comprising a third selection marker and an internal ribosome entry site (IRES), wherein the IRES is operably linked to the third selection marker.
 91. The TI host cell of claim 88, further comprising a third RRS, wherein the third RRS is located between the first and the second RRS, and the third RRS is heterospecific relative the first or the second RRS.
 92. The TI host cell of claim 85, wherein the exogenous nucleotide sequence comprises at least one selection marker and at least one exogenous sequence of interest (SOI).
 93. The TI host cell claim 85, wherein the exogenous nucleotide sequence further comprises at least one exogenous SOI.
 94. The TI host cell of claim 93, wherein the exogenous SOI is located between the first and the second RRS.
 95. The TI host cell of claim 93, wherein the SOI encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc fusion protein.
 96. The TI host cell of claim 85, wherein the exogenous nucleotide sequence further comprises at least one exogenous SOI located between the first and the third RRS, and at least one exogenous SOI located between the third and the second RRS.
 97. The TI host cell of claim 96, wherein the SOI encodes a single chain antibody, an antibody light chain, an antibody heavy chain, a single-chain Fv fragment (scFv), or an Fc fusion protein.
 98. A method of preparing a TI host cell expressing a polypeptide of interest comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus of the genome of the TI host cell, wherein the locus is at least about 90% homologous to a sequence selected from contig NW_006874047.1, contig NW_006884592.1, contig NW_006881296.1, contig NW_003616412.1, contig NW_003615063.1, contig NW_006882936.1, and contig NW_003615411.1, wherein the exogenous nucleotide sequence comprises two RRSs, flanking at least one first selection marker; b) introducing into the cell provided in a) a vector comprising two RRSs matching the two RRSs on the integrated exogenous nucleotide sequence and flanking at least one exogenous SOI and at least one second selection marker; c) introducing a recombinase or a nucleic acid encoding a recombinase, wherein the recombinase recognizes the RRSs; and d) selecting for TI cells expressing the second selection marker to thereby isolate a TI host cell expressing the polypeptide.
 99. A method for expressing a polypeptide of interest comprising: a) providing a TI host cell comprising at least one exogenous SOI and at least one selection marker where the at least one exogenous SOI and at least one selection marker are flanked by two RRSs integrated within a locus of the genome of the TI host cell, wherein the locus is at least about 90% homologous to a sequence selected from: contig NW_006874047.1, contig NW_006884592.1, contig NW_006881296.1, contig NW_003616412.1, contig NW_003615063.1, contig NW_006882936.1, and contig NW_003615411.1; and b) culturing the cell in a) under conditions suitable for expressing the polypeptide of interest and recovering a polypeptide of interest therefrom.
 100. The method of claim 99, further comprising a second polypeptide of interest comprising: a) providing a TI host cell comprising an exogenous nucleotide sequence integrated at a site within a locus of the genome of the host cell, wherein the locus is at least about 90% homologous to a sequence selected from contig NW_006874047.1, contig NW_006884592.1, contig NW_006881296.1, contig NW_003616412.1, contig NW_003615063.1, contig NW_006882936.1, and contig NW_003615411.1, wherein the exogenous nucleotide sequence comprises a first and a second RRS flanking at least one first selection marker, and a third RRS located between the first and the second RRS, and all the RRSs are heterospecific; b) introducing into the cell provided in a) a first vector comprising two RRSs matching the first and the third RRS on the integrated exogenous nucleotide sequence and flanking at least one first exogenous SOI and at least one second selection marker; c) introducing into the cell provided in a) a second vector comprising two RRSs matching the second and the third RRS on the integrated exogenous nucleotide sequence and flanking at least one second exogenous SOI; d) introducing one or more recombinases, or one or more nucleic acids encoding one or more recombinases, wherein the one or more recombinases recognize the RRSs; and e) selecting for TI cells expressing the second selection marker to thereby isolate a TI host cell expressing the first and second polypeptides of interest.
 101. A vector comprising two nucleotide sequences at least 50% homologous to a. two reference sequences selected from any portion of contig NW_006874047.1; b. two reference sequences selected from any portion of contig NW_006884592.1; c. two reference sequences selected from any portion of contig NW_006881296.1; d. two reference sequences selected from any portion of contig NW_003616412.1; e. two reference sequences selected from any portion of contig NW_003615063.1; f. two reference sequences selected from any portion of contig NW_006882936.1; or g. two reference sequences selected from any portion of contig NW_003615411.1, wherein said sequences flank a DNA cassette, and wherein the DNA cassette comprises at least one selection marker and at least one exogenous SOI flanked by two RRSs. 